Torsten Neuhof

Nonribosomal Peptide Synthesis in Schizosaccharomyces pombe and the Architectures of Ferrichrome-Type Siderophore Synthetases in Fungi

A nonribosomal peptide synthetase (NRPS) in Schizosaccharomyces pombe, which possesses an unusual structure incorporating three adenylation domains, six thiolation domains and six condensation domains, has been shown to produce the cyclohexapeptide siderophore ferrichrome. One of the adenylation domains is truncated and contains a distorted key motif. Substrate‐binding specificities of the remaining two domains were assigned by molecular modelling to glycine and to N‐acetyl‐N‐hydroxy‐L‐ornithine. Hexapeptide siderophore synthetase genes of Magnaporthe grisea and Fusarium graminearum were both identified and analyzed with respect to substrate‐binding sites, and the predicted product ferricrocin was identified in each. A comparative analysis of these synthetase systems, including those of the basidiomycete Ustilago maydis, the homobasidiomycete Omphalotus olearius and the ascomycetes Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, Cochliobolus heterostrophus, Neurospora crassa and Aureobasidium pullulans, revealed divergent domain compositions with respect to their number and positioning, although all produce similar products by iterative processes. A phylogenetic analysis of both NRPSs and associated L‐N5‐ornithine monooxygenases revealed that ferrichrome‐type siderophore biosynthesis has coevolved in fungi with varying in trans interactions of NRPS domains.

Direct identification of hydrophobins and their processing in Trichoderma using intact-cell MALDI-TOF MS.

Intact‐cell MS (ICMS) was applied for the direct detection of hydrophobins in various species and strains of Hypocrea/Trichoderma. In both mycelia and spores, dominating peaks were identified as hydrophobins by detecting mass shifts of 8 Da of reduced and unreduced forms, the analysis of knockout mutants, and comparison with protein databases. Strain‐specific processing was observed in the case of Hypocrea jecorina (anamorph Trichoderma reesei). An analysis of 32 strains comprising 29 different species of Trichoderma and Hypocrea showed hydrophobin patterns that were specific at both at the species and isolate (subspecies) levels. The method therefore permits rapid and direct detection of hydrophobin class II compositions and may also provide a means to identify Trichoderma (and other fungal) species and strains from microgram amounts of biomass without prior cultivation.

Novel Hydrophobins from Trichoderma Define a New Hydrophobin Subclass: Protein Properties, Evolution, Regulation and Processing

Hydrophobins are small proteins, characterised by the presence of eight positionally conserved cysteine residues, and are present in all filamentous asco- and basidiomycetes. They are found on the outer surfaces of cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment. Hydrophobins are conventionally grouped into two classes (class I and II) according to their solubility in solvents, hydropathy profiles and spacing between the conserved cysteines. Here we describe a novel set of hydrophobins from Trichoderma spp. that deviate from this classification in their hydropathy, cysteine spacing and protein surface pattern. Phylogenetic analysis shows that they form separate clades within ascomycete class I hydrophobins. Using T. atroviride as a model, the novel hydrophobins were found to be expressed under conditions of glucose limitation and to be regulated by differential splicing.

The Production of Multiple Small Peptaibol Families by Single 14‐Module Peptide Synthetases in Trichoderma/Hypocrea

The most common sequences of peptaibiotics are 11‐residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14‐residue peptaibols sharing partial sequence identity. Genome sequencing projects of three Trichoderma strains of the major clades reveal the presence of up to three types of nonribosomal peptide synthetases with 7, 14, or 18–20 amino acid‐adding modules. Here, we provide evidence that the 14‐module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina), and T. atroviride produces both 11‐ and 14‐residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid‐activating domains and modules. The sequences of these peptides may be predicted from the gene sequences and have been confirmed by analysis of families of 11‐ and 14‐residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 sequences determined, 2 new), and the recently established trichovirins A from T. virens. The distribution of 11‐ and 14‐residue products is strain‐specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.

Formation of Atroviridin by Hypocrea atroviridis Is Conidiation Associated and Positively Regulated by Blue Light and the G Protein GNA3

ABSTRACT Species of the mycoparasitic fungal genus Hypocrea/Trichoderma are prominent producers of peptaibols, a class of small linear peptides of fungal origin. Some of these peptaibols have been shown to act synergistically with cell-wall-degrading enzymes in the inhibition of the growth of other fungi in vitro and in vivo. Here we present the structure of the Hypocrea atroviridis peptaibol synthetase gene (pbs1), deduced from the genome sequence of H. atroviridis. It consists of 19 typical peptide synthetase modules with the required additional modifying domains at the N and C termini. Phylogenetic and similarity analyses of the individual amino acid-activating modules is consistent with its ability to synthesize atroviridins. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of surface-grown cultures of H. atroviridis showed that no peptaibols were formed during vegetative growth, but a microheterogenous mixture of atroviridins accumulated when the colonies started to sporulate. This correlation between sporulation and atroviridin formation was shown to be independent of the pathway inducing sporulation (i.e., light, mechanical injury and carbon starvation, respectively). Atroviridin formation was dependent on the function of the two blue light regulators, BLR1 and BLR2, under some but not all conditions of sporulation and was repressed in a pkr1 (regulatory subunit of protein kinase A) antisense strain with constitutively active protein kinase A. Conversely, however, loss of function of the Gα-protein GNA3, which is a negative regulator of sporulation and leads to a hypersporulating phenotype, fully impairs atroviridin formation. Our data show that formation of atroviridin by H. atroviridis occurs in a sporulation-associated manner but is uncoupled from it at the stage of GNA3.

Differential Regulation and Posttranslational Processing of the Class II Hydrophobin Genes from the Biocontrol Fungus Hypocrea atroviridis

ABSTRACT Hydrophobins are small extracellular proteins, unique to and ubiquitous in filamentous fungi, which mediate interactions between the fungus and environment. The mycoparasitic fungus Hypocrea atroviridis has recently been shown to possess 10 different class II hydrophobin genes, which is a much higher number than that of any other ascomycete investigated so far. In order to learn the potential advantage of this hydrophobin multiplicity for the fungus, we have investigated their expression patterns under different physiological conditions (e.g., vegetative growth), various conditions inducing sporulation (light, carbon starvation, and mechanical injury-induced stress), and confrontation with potential hosts for mycoparasitism. The results show that the 10 hydrophobins display different patterns of response to these conditions: one hydrophobin (encoded by hfb-2b) is constitutively induced under all conditions, whereas other hydrophobins were formed only under conditions of carbon starvation (encoded by hfb-1c and hfb-6c) or light plus carbon starvation (encoded by hfb-2c, hfb-6a, and hfb-6b). The hydrophobins encoded by hfb-1b and hfb-5a were primarily formed during vegetative growth and under mechanical injury-provoked stress. hfb-22a was not expressed under any conditions and is likely a pseudogene. None of the 10 genes showed a specific expression pattern during mycoparasitic interaction. Most, but not all, of the expression patterns under the three different conditions of sporulation were dependent on one or both of the two blue-light regulator proteins BLR1 and BLR2, as shown by the use of respective loss-of-function mutants. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of mycelial solvent extracts provided sets of molecular ions corresponding to HFB-1b, HFB-2a, HFB-2b, and HFB-5a in their oxidized and processed forms. These in silico-deduced sequences of the hydrophobins indicate cleavages at known signal peptide sites as well as additional N- and C-terminal processing. Mass peaks observed during confrontation with plant-pathogenic fungi indicate further proteolytic attack on the hydrophobins. Our study illustrates both divergent and redundant functions of the 10 hydrophobins of H. atroviridis.

Dipeptide synthesis by an isolated adenylate-forming domain of non-ribosomal peptide synthetases (NRPS)

A deletion mutant of tyrocidine synthetase 1 (ΔΔTY1), comprising the adenylation domain of TY1 as an independent functional adenylate‐forming unit, was used to investigate the ability of the adenylation domain in non‐ribosomal peptide synthetases to catalyse peptide bond formation from the aminoacyl adenylate intermediate. The results demonstrate that only one substrate amino acid needs to be activated as an aminoacyl adenylate. In view of the potential exploitation of peptide synthetases for enzymatic synthesis of dipeptides of choice, it is important to note that this does not necessarily require a dimodular construct or an intermediate acyl transfer step.

Penicillin biosynthesis: intermediates of biosynthesis of δ-l-α-aminoadipyl-l-cysteinyl-d-valine formed by ACV synthetase from Acremonium chrysogenum

The tripeptide δ‐l‐α‐aminoadipyl‐l‐cysteinyl‐d‐valine (LLD‐ACV) is synthesised by the multifunctional enzyme ACV synthetase integrating four steps of the penicillin and cephalosporin biosynthetic pathway. Peptide synthesis follows the thiotemplate mechanism from intermediates bound as thioesters to the enzyme. The formation of δ‐(l‐α‐aminoadipyl)‐l‐cysteinyl‐thioester in the absence of l‐valine was shown by isolation of the enzyme–substrate complex and cleavage of the covalently bound intermediate with performic acid. The dipeptide was recovered as cysteic acid or cysteic acid oxime and detected by HPLC and MALDI‐TOF mass spectrometry. We conclude that the first peptide bond is formed between δ‐carboxyl of l‐aminoadipic acid and l‐cysteine, followed by addition of the dipeptidyl intermediate to l‐valine.