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.

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The hypersecreting mutant Trichoderma reesei RUT-C30 (ATCC 56765) is one of the most widely used strains of filamentous fungi for the production of cellulolytic enzymes and recombinant proteins, and for academic research. The strain was obtained after three rounds of random mutagenesis of the wild-type QM6a in a screening program focused on high cellulase production and catabolite derepression. Whereas RUT-C30 achieves outstanding levels of protein secretion and high cellulolytic activity in comparison to the wild-type QM6a, recombinant protein production has been less successful. Here, we bring together and discuss the results from biochemical-, microscopic-, genomic-, transcriptomic-, glycomic-and proteomic-based research on the RUT-C30 strain published over the last 30 years.

The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes.

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Lateral gene transfer has been proposed as a fundamental process underlying bacterial diversity. Transposons, plasmids and phage are widespread and have been shown to significantly contribute to lateral gene transfer. However, the processes by which disparate genes are assembled and integrated into the host regulatory network to yield new phenotypes are poorly known. Recent discoveries about the integron/gene cassette system indicate it has the potential to play a role in this process. Gene cassettes are small mobile elements typically consisting of a promoterless orf and a recombination site. Integrons are capable of acquisition and re-arrangement of gene cassettes and of the expression of their associated genes. The potential of the integron/gene cassette system is thus largely determined by the diversity contained within the cassette pool and the rate at which integrons sample this pool. We show here using a polymerase chain reaction (PCR) approach by which the environmental gene cassette (EGC) metagenome can be directly sampled that this metagenome contains both protein-coding and non-protein coding genes. Environmental gene cassette-associated recombination sites showed greater diversity than previously seen in integron arrays. Class 1 integrons were shown to be capable of accessing this gene pool through tests of recombinational activity with a representative range of EGCs. We propose that gene cassettes represent a vast, prepackaged genetic resource that could be thought of as a metagenomic template for bacterial evolution.

Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solidphase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known. Keywords : cellobiohydrolase I; cellulase; fungal glycosylation; Trichoderma; N-acetylglucosamine.Cellobiohydrolase I (CBHI) is the major family C glycoprotein secreted by the filamentous fungus Trichoderma reesei, where it operates in synergy with other exo-glucanases and endo-glucanases to achieve the degradation of cellulose [1]. CBHI is typically secreted in the order of several grams/litre, and many aspects of its structure and biology are well characterised. Like most other fungal cellulolytic enzymes characterised, CBHI conforms to a well-defined multi-domain structure, typified by a relatively large catalytic (core) domain spatially removed from a smaller cellulose-binding domain (CBD) by a highly O-glycosylated linker domain [2]. Presumably, the CBD serves as a form of anchor, and the linker a form of torsional leash, providing the catalytic region with access to a wide range of potential (and perhaps otherwise inaccessible) sites [3]. Although the catalytic domain of CBHI by itself is sufficient for cellulose binding and full catalytic activity at low enzyme to cellulose ratios, a functional CBD with sufficient spatial separation from the catalytic region is necessary for cellulolytic activity at higher enzyme-to-cellulose ratios [3]. The importance of the linker domain in maintaining spatial orientation of catalytic Correspondence to N. H. Packer,

Glycosylation of proteins is important for protein stability, secretion, and localization. In this study, we have investigated the glycan synthesis pathways of 12 filamentous fungi including those of medical/agricultural/industrial importance for which genomes have been recently sequenced. We have adopted a systems biology approach to combine the results from comparative genomics techniques with high confidence information on the enzymes and fungal glycan structures, reported in the literature. From this, we have developed a composite representation of the glycan synthesis pathways in filamentous fungi (both N- and O-linked). The N-glycosylation pathway in the cytoplasm and endoplasmic reticulum was found to be highly conserved evolutionarily across all the filamentous fungi considered in the study. In the final stages of N-glycan synthesis in the Golgi, filamentous fungi follow the high mannose pathway as in Saccharomyces cerevisiae, but the level of glycan mannosylation is reduced. Highly specialized N-glycan structures with galactofuranose residues, phosphodiesters, and other insufficiently trimmed structures have also been identified in the filamentous fungi. O-Linked glycosylation in filamentous fungi was seen to be highly conserved with many mannosyltransferases that are similar to those in S. cerevisiae. However, highly variable and diverse O-linked glycans also exist. We have developed a web resource for presenting the compiled data with user-friendly query options, which can be accessed at www.fungalglycans.org. This resource can assist attempts to remodel glycosylation of recombinant proteins expressed in filamentous fungal hosts.