Intact-cell MALDI-TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma/Hypocrea: can molecular phylogeny of species predict peptaibol structures?

Abstract

Peptaibols are characteristic linear a-aminoisobutyrate-containing peptides produced by certain Ascomycetes, especially of the genus Hypocrea/Trichoderma [Hypocrea and Trichoderma are the names for the teleo-and anamorph forms of the same taxon; where known to occur in nature, the teleomorph is used to name the species. To aid the inexperienced reader, both names (the less well known one in parentheses) are given at the first mention of each species.] Here we have investigated whether phylogenetic relationships within Trichoderma permit a prediction of the peptaibol production profiles. To this end, representative strains from a third (28) of the known species of Trichoderma, identified by the sequences of diagnostic genes and covering most clades of the established multilocus phylogeny of Trichoderma/Hypocrea, were investigated by intact-cell MALDI-TOF mass spectrometry. Peptaibols were detected in all strains, and some strains were found to produce up to five peptide families of different sizes. Comparison of the data with phylogenies derived from rRNA spacer regions (ITS1 and 2) and RNA polymerase subunit B (rpb2) gene sequences did not show a strict correlation with the types and sequences of the peptaibols produced, but the production of some groups of peptaibols appears to be found only in some clades or sections of the genus, which could be used for more targeted screening of novel compounds of this type. In an analysis of peptaibol structures, we have defined conserved key positions and have further identified and compared sequences of the corresponding adenylate domains within non-ribosomal peptide synthetases producing trichovirins, paracelsins and atroviridins. These phylogenies are not concordant with those of their producers Hypocrea virens, Hypocrea jecorina and Hypocrea atroviridis as obtained from ITS1 and 2, and rpb2, respectively, and therefore hint at a complex history of peptaibol diversity.

Two Classes of New Peptaibols Are Synthesized by a Single Non-ribosomal Peptide Synthetase of Trichoderma virens

“…Depending upon the strain, Trichoderma spp. produce either a 11-residue peptaibol or a 14-residue peptaibol, or both (19). Although, we initially reported that the deletion of Tex1 resulted in the loss of all three (18, 14, and 11-residue) peptaibols, it was subsequently confirmed by us and another group that Tex1 codes for only the 18-residue peptaibol (26,33,34).…”

“…As already suggested, both types of peptaibols could originate from a single peptide synthetase. Based on the available sequence comparison of 11-and 14-residue peptaibols, Neuhof et al (19) predicted that SF4 11-(group 6) and 14-residue (groups 8, 9, and 11) compounds could be produced by a single peptaibol synthetase by deletion of modules 3-5 (Leu3-Pro4-Aib5). Nevertheless, taking into account the alignments of the sequences, it seems that in Gv29-8, 11-residue peptaibols could be derived from 14-residue peptaibols by internal deletion of the residues in positions 4, 5, and 6 or could be synthesized by an enzyme that is lacking the modules for incorporation of residues 4, 5, and 6.…”

“…The first genetic evidence of the involvement of an NRPS in peptaibol synthesis came from our studies of Tex1, where we demonstrated that an 18-module NRPS synthesizes the largest peptaibol produced by T. virens (26,33,34). However, so far, there have been no genetic studies on the synthesis of the shorter peptaibols, even though presence of these peptaibols are reported in many Trichoderma/Hypocrea spp (19). In this study, we present many new sequences of the 11-and 14-residue peptaibols of T. virens, and for the first time, provide genetic evidence that a single 14-module NRPS is responsible for the synthesis of these two classes of peptaibols.…”

“…Subfamily 4 (SF4) is comprised of peptides of 11-14 residues, also sharing sequence similarities, but having no sequence relationship to SF1. Subfamilies 5 and 9 (SF5 and -9) have only a few members and comprise peptides with 11 or 6 and 7 residues, respectively, again with no sequence similarities to the other subfamilies (19). An extensive survey of 28 Trichoderma/Hypocrea species for peptaibol production by intact-cell MALDI-TOF mass spectrometry (ICMS) revealed that peptaibols are produced by all strains with some producing up to five peptide families of different sizes (19).…”

“…Subfamilies 5 and 9 (SF5 and -9) have only a few members and comprise peptides with 11 or 6 and 7 residues, respectively, again with no sequence similarities to the other subfamilies (19). An extensive survey of 28 Trichoderma/Hypocrea species for peptaibol production by intact-cell MALDI-TOF mass spectrometry (ICMS) revealed that peptaibols are produced by all strains with some producing up to five peptide families of different sizes (19). In addition to terrestrial strains, peptaibols have also been isolated from marine strains of Trichoderma (7,20,22,23).…”