Sequences of stilboflavin C: towards the peptaibiome of the filamentous fungus Stilbella (= Trichoderma) flavipes.

Filamentous fungi of the genus Stilbella are recognized as an abundant source of naturally occurring α-aminoisobutyric acid-containing peptides. The culture broth of Stilbella (Trichoderma) flavipes CBS 146.81 yielded a mixture of peptides named stilboflavins (SF), and these were isolated and separated by preparative TLC into groups named SF-A, SF-B, and SF-C. Although all three of these groups resolved as single spots on thin-layer chromatograms, HPLC analysis revealed that each of the groups represents very microheterogeneous mixtures of closely related peptides. Here, we report on the sequence analysis of SF-C peptides, formerly isolated by preparative TLC. HPLC coupled to QqTOF-ESI-HRMS provided the sequences of 10 16-residue peptides and five 19-residue peptides, all of which were N-terminally acetylated. In contrast to the previously described SF-A and SF-B peptaibols, SF-C peptaibols contain Ser-Alaol or Ser-Leuol, which are rarely found as C-termini, and repetitive Leu-Aib-Gly sequences, which have not been detected in peptaibols before. Taking the previously determined sequences of SF-A and SF-B into account, the entirety of peptides produced by S. flavipes (the 'peptaibiome') approaches or exceeds 100 non-ribosomally biosynthesized peptaibiotics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Peptaibol, secondary-metabolite, and hydrophobin pattern of commercial biocontrol agents formulated with species of the Trichoderma harzianum complex.

The production of bioactive polypeptides (peptaibiotics) in vivo is a sophisticated adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species for colonizing and defending their natural habitats. This feature is of major practical importance, as the detection of peptaibiotics in plant-protective Trichoderma species, which are successfully used against economically relevant bacterial and fungal plant pathogens, certainly contributes to a better understanding of these complex antagonistic interactions. We analyzed five commercial biocontrol agents (BCAs), namely Canna(®) , Trichosan(®) , Vitalin(®) , Promot(®) WP, and TrichoMax(®) , formulated with recently described species of the Trichoderma harzianum complex, viz. T. afroharzianum, T. simmonsii, and T. guizhouense. By using the well-established, HPLC/MS-based peptaibiomics approach, it could unequivocally be demonstrated that all of these formulations contained new and recurrent peptaibols, i.e., peptaibiotics carrying an acetylated N-terminus, the C-terminus of which is reduced to a 1,2-amino alcohol. Their chain lengths, including the amino alcohol, were 11, 14, and 18 residues, respectively. Peptaibols were also to be the dominating secondary metabolites in plate cultures of the four strains obtained from four of the Trichoderma- based BCAs, contributing 95% of the UHPLC-UV/VIS peak areas and 99% of the total ion count MS peak area from solid media. Furthermore, species-specific hydrophobins, as well as non-peptaibiotic secondary metabolites, were detected, the latter being known for their antifungal, siderophore, or plant-growth-promoting activities. Notably, none of the isolates produced low-molecular weight mycotoxins.

Front line defenders of the ecological niche! Screening the structural diversity of peptaibiotics from saprotrophic and fungicolous Trichoderma/Hypocrea species.

Approximately 950 individual sequences of non-ribosomally biosynthesised peptides are produced by the genus Trichoderma/Hypocrea that belong to a perpetually growing class of mostly linear antibiotic oligopeptides, which are rich in the non-proteinogenic α-aminoisobutyric acid (Aib). Thus, they are comprehensively named peptaibiotics. Notably, peptaibiotics represent ca. 80 % of the total inventory of secondary metabolites currently known from Trichoderma/Hypocrea. Their unique membrane-modifying bioactivity results from amphipathicity and helicity, thus making them ideal candidates in assisting both colonisation and defence of the natural habitats by their fungal producers. Despite this, reports on the in vivo-detection of peptaibiotics have scarcely been published in the past. In order to evaluate the significance of peptaibiotic production for a broader range of potential producers, we screened nine specimens belonging to seven hitherto uninvestigated fungicolous or saprotrophic Trichoderma/Hypocrea species by liquid chromatography coupled to electrospray high resolution mass spectrometry. Sequences of peptaibiotics found were independently confirmed by analysing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens. Of the nine species examined, five were screened positive for peptaibiotics. A total of 78 peptaibiotics were sequenced, 56 (=72 %) of which are new. Notably, dihydroxyphenylalaninol and O-prenylated tyrosinol, two C-terminal residues, which have not been reported for peptaibiotics before, were found as well as new and recurrent sequences carrying the recently described tyrosinol residue at their C-terminus. The majority of peptaibiotics sequenced are 18- or 19-residue peptaibols. Structural homologies with 'classical representatives' of subfamily 1 (SF1)-peptaibiotics argue for the formation of transmembrane ion channels, which are prone to facilitate the producer capture and defence of its substratum.

Screening the biosphere: the fungicolous fungus Trichoderma phellinicola, a prolific source of hypophellins, new 17-, 18-, 19-, and 20-residue peptaibiotics.

To investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus Trichoderma phellinicola (syn. Hypocrea phellinicola) growing on its natural host Phellinus ferruginosus. Results revealed that a particular group of non-ribosomal antibiotic polypeptides, peptaibiotics, which contain the non-proteinogenic marker amino acid, α-aminoisobutyric acid, was biosynthesized in the natural habitat by the fungicolous producer and, consequently, released into the host. By means of liquid chromatography coupled to electrospray high-resolution time-of-flight mass spectrometry, we detected ten 20-residue peptaibols in the specimen. Sequences of peptaibiotics found in vivo were independently confirmed by analyzing the peptaibiome of an agar plate culture of T. phellinicola CBS 119283 (ex-type) grown under laboratory conditions. Notably, this strain could be identified as a potent producer of 39 new 17-, 18-, and 19-residue peptaibiotics, which display the same building scheme as the 20-residue peptaibols found in the specimen. Two of the 19-residue peptaibols are tentatively assigned to carry tyrosinol, a novel C-terminal residue, as deduced from high-resolution tandem mass-spectrometry data. For the new peptaibiotics produced by T. phellinicola, the name 'hypophellin(s)', based on the teleomorph name, is introduced.

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.

Fungal cyclooligomer depsipeptides: from classical biochemistry to combinatorial biosynthesis.

This review surveys the biological activities and the iterative and recursive biosynthetic mechanisms of fungal cyclooligomer depsipeptides, and their structural diversification by various combinatorial biosynthetic methods.

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.

A survey of nonribosomal peptide synthetase (NRPS) genes in Aspergillus nidulans.

The genome of Aspergillus nidulans carries 27 genes encoding nonribosomal peptide synthetase (NRPS) structures, although only five of these forming peptides and amino acid containing metabolites have been identified so far. This manuscript describes domain structures, substrate binding pockets and related genes and gene clusters and summarizes our current state of product prediction of fungal NRPS systems.

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.

Differential regulation and posttranslational processing of the class II hydrophobin genes from the biocontrol fungus Hypocrea atroviridis.

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.

MALDI-TOF-MS detection of the low molecular weight neurotoxins anatoxin-a and homoanatoxin-a on lyophilized and fresh filaments of axenic Oscillatoria strains.

Anatoxin-a (ANTX) and homoanatoxin-a (HANTX) are low molecular weight neurotoxic secondary amines of 165 and 179 Da, respectively. We applied matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the detection of ANTX and HANTX directly on lyophilized and fresh filaments of axenic strains of the genus Oscillatoria, using 2,5-dihydroxybenzoic acid as matrix and purified ANTX and HANTX as references. To counteract the span of low molecular mass ions (< m/z 1000) generated by the matrix, we induced the matrix-suppression effect to obtain high quality ANTX/HANTX MALDI signals. MALDI desorption/ionization of the matrix-ANTX and the matrix-HANTX generated protonated molecules [M+H](+) at m/z 166.12322 and 180.1372, respectively. The masses obtained from the analysis of lyophilized filaments of the ANTX-producer Oscillatoria sp. strain PCC 9240 (m/z 166.15) and of fresh filaments of the HANTX-producers Oscillatoria sp. strains PCC 6506 (m/z 180.1375), PCC 9029 (m/z 180.1334) and PCC 10111 (m/z 180.13996) corresponded to the protonated molecular ions of ANTX and HANTX, respectively. Therefore, the application of MALDI-TOF-MS for the detection of cyanobacterial anatoxins in clonal and axenic strains of the cyanobacterial culture collections worldwide may help to assess ANTX/HANTX incidence among cyanobacteria.

Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated by blue light and the G protein GNA3.

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 Galpha-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.

Recent advances and future prospects in peptaibiotics, hydrophobin, and mycotoxin research, and their importance for chemotaxonomy of Trichoderma and Hypocrea.

Fungi of the genus Trichoderma with teleomorphs in Hypocrea are abundant producers of a group of amphiphilic, non-ribosomal peptide antibiotics, which are rich in the non-proteinogenic amino acid Aib (alpha-aminoisobutyric acid). They are referred to as peptaibiotics, or peptaibols, if a 1,2-amino alcohol is present at the C-terminus. Trichoderma/Hypocrea, like other ascomycetous fungi, also produce hydrophobins, a class of small, cysteine-rich proteins. Advanced soft ionization mass spectrometric techniques such as LC-CID-MS, LC-ESI-MS(n), and IC-MALDI-TOF-MS enabled the high-throughput analysis, simultaneous detection and sequence determination of peptaibiotics and hydrophobins from minute quantities of fungal materials. Some Trichoderma species have been recognized to produce peptaibiotics as well as simple mycotoxins of the trichothecene group. The combination of sequence data of both groups of peptides with the pattern of low-molecular-weight secondary metabolites, including trichothecene-type mycotoxins, independently confirmed the results of morphological, molecular, and phylogenetic analyses. This approach established a new lineage in Trichoderma/Hypocrea, the Brevicompactum clade, comprising four new and one redescribed species. Notably, commercial preparations of single or mixed cultures of Trichoderma species, in particular T. harzianum, and T. koningii, are registered as biocontrol agents for soil and plant pathogens. In this context, it is emphasized that the four mycotoxin-producing species of the recently established Brevicompactum clade (T. brevicompactum, T. arundinaceum, T. turrialbense, and T. protrudens) are not closely related to any of the Trichoderma species currently used as biocontrol agents. Furthermore, possible health concerns about release of peptaibiotics in the biosphere are discussed with respect to their bioactivities and their use as drugs in human and veterinary medicine. Finally, future prospects regarding novel bioactivities and further research needs, including interdisciplinary taxonomic approaches, are outlined.

Cyanobacterial peptides - nature's own combinatorial biosynthesis.

Cyanobacterial secondary metabolites have attracted increasing scientific interest due to bioactivity of many compounds in various test systems. Among the known structures, oligopeptides are often found with many congeners sharing conserved substructures, while being highly variable in others. A major part of known oligopeptides are of non-ribosomal origin and can be grouped into classes with conserved structural properties. Thus, the overall structural diversity of cyanobacterial oligopeptides only seemingly suggests an equally high diversity of biosynthetic pathways and respective genes. For each class of peptides, some of which have been found in all major branches of the cyanobacterial evolutionary tree, homologous synthetases and genes can be inferred. This implies that non-ribosomal peptide synthetase genes are a very ancient part of the cyanobacterial genome and presumably have evolved by recombination and duplication events to reach the present structural diversity of cyanobacterial oligopeptides. In addition, peptide synthetases would appear to be an essential part of the cyanobacterial evolution and physiology. The present review presents an overview of the biosynthesis of cyanobacterial peptides and corresponding gene clusters, the structural diversity of structural types and structural variations within peptide classes, and implications for the evolution and plasticity of biosynthetic genes and the potential function of cyanobacterial peptides.

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.

Peptaibol production by sepedonium strains parasitizing boletales.

Fungi of the genus Sepedonium (anamorphic ascomycetes) are known to infect fruiting bodies of Basidiomycetes of the order Boletales. We have characterized twelve Sepedonium isolates by intact-cell mass spectrometry (IC-MS) with the help of respective biomarkers and their metabolite spectra focusing on peptaibol production. A strain of mycoparasitic S. chalcipori was grown in solid-state fermentation, and tylopeptin production was found, suggesting an ascomycete origin of these peptaibols, which were first described in the basidiomycete Tylopilus neofelleus. In addition, the structures of two new peptaibols, chalciporin A (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Ser-Leu-Ala-Leu-Aib-Gln-Leuol) and chalciporin B (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Gln-Aib-Ala-Leu-Aib-Gln-Leuol) are presented. The IC-MS technique was applied for in situ peptaibol analysis of Sepedonium strains growing on Boletales, in particular S. chrysospermum infecting Xerocomus cf. badius. We found chrysospermins at the surface and within basidiomycete tissue, as well as in the cultivated parasite.

Detection and identification of oligopeptides in Microcystis (cyanobacteria) colonies: toward an understanding of metabolic diversity.

Cyanobacteria and particularly Microcystis sp. (Chroococcales) are known to produce a multitude of peptide metabolites. Here we report on the mass spectral analysis of cyanobacterial peptides in individual colonies of Microcystis sp. collected in a drinking water reservoir. A total number of more than 90 cyanopeptides could be detected, 61 of which could be identified either as known peptides or new structural variants of known peptide classes. For 18 new peptides flat structures are proposed. New congeners differed from known ones mainly in chlorination (aeruginosins), methylation (microginins), or amino acid sequences (cyanopeptolins). The high number of peptides and especially the new peptides underline the capability of Microcystis strains as producers of a high diversity of potentially bioactive compounds.

ATPase activity of non-ribosomal peptide synthetases.

Adenylation domains of non-ribosomal peptide synthetases (NRPS) catalyse the formation of aminoacyl adenylates, and in addition synthesize mono- and dinucleoside polyphosphates. Here, we show that NRPS systems furthermore contain an ATPase activity in the range of up to 2 P(i)/min. The hydrolysis rate by apo-tyrocidine synthetase 1 (apo-TY1) is enhanced in the presence of non-cognate amino acid substrates, correlating well with their structural features and the diminishing adenylation efficiency. A comparative analysis of the functional relevance of an analogous sequence motif in P-type ATPases and adenylate kinases (AK) allowed a putative assignment of the invariant aspartate residue from the TGDLA(V)R(K) core sequence in NRPS as the Mg(2+) binding site. Less pronounced variations in ATPase activity are observed in domains with relaxed amino acid specificity of gramicidin S synthetase 2 (GS2) and delta-(L-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), known to produce a set of substitutional variants of the respective peptide product. These results disclose new perspectives about the mode of substrate selection by NRPS.

Nonribosomal peptide synthetases-evidence for a second ATP-binding site.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) catalyses, via the protein thiotemplate mechanism, the nonribosomal biosynthesis of the penicillin and cephalosporin precursor tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV). The complete and fully saturated biosynthetic system approaches maximum rate of product generation with increasing ATP concentration. Nonproductive adenylation of ACVS, monitored utilising the ATP-[32P]PP(i) exchange reaction, has revealed substrate inhibition with ATP. The kinetic inhibition pattern provides evidence for the existence of a second nucleotide-binding site with possible implication in the regulatory mechanism. Under suboptimal reaction conditions, in the presence of MgATP(2-), L-Cys and inorganic pyrophosphatase, ACVS forms adenosine(5')tetraphospho(5')adenosine (Ap(4)A) from the reverse reaction of adenylate formation involving a second ATP molecule. The potential location of the second ATP binding site was deduced from sequence comparisons and molecular visualisation in conjunction to data obtained from biochemical analysis.