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1 TU Berlin, Institut für Chemie, FG Biochemie und Molekulare Biologie, Franklinstr. 29, 10587 Berlin, Germany
2 Forschungsbereich Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, TU Wien, Getreidemarkt 9-166, 1060 Wien, Austria
Correspondence
Hans von Döhren
Doehren{at}chem.tu-berlin.de
Christian Kubicek
ckubicek{at}mail.zserv.tuwien.ac.at
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-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.
-aminoisobutyric acid; ICMS, intact-cell MALDI-TOF mass spectrometry; NRPS, non-ribosomal peptide synthetase; SFn, subfamily n
Present address: Anagnostec GmbH, Im Biotechnologiepark TGZ II, 14943 Luckenwalde, Germany.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Whitmore & Wallace, 2004). These linear, non-ribosomally formed peptides of 5–20 residues are characteristically highly abundant in -aminoisobutyric acid (Aib), and contain an N-acyl (usually acetyl) terminus and a C-terminal alcohol, such as phenylalaninol or leucinol (Benedetti et al., 1982; Brückner & Graf, 1983; Brückner et al., 1984). A database of peptaibol structures (www.cryst.bbk.ac.uk/peptaibol/home.shtml) currently lists more than 300 structures, grouped into nine distinct subfamilies, which can be further differentiated according to their amino acid number and substitution pattern, and terminal processing (Chugh & Wallace, 2001). Peptaibols/peptaibiotics are also formed by other fungi of the Hypocreales, but reports of their production by basidiomycetes seem to have been due to infections of the basidiomycte fruiting bodies by these fungi which are frequently mycoparasites (Degenkolb et al., 2003; Neuhof et al., 2007b). Within the genus Trichoderma/Hypocrea, so far subfamilies 1, 4, 5 and 9 have been described (Szekeres et al., 2005). Subfamily 1 (SF1) comprises about half of the known structures and combines peptides ranging from 18 to 20 aa in length. All these peptides have partial sequence identities or similarities. Subfamily 4 (SF4) combines peptides of 11 or 14 aa, also sharing sequence similarities, but have 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, again with no similarities to the other subfamilies.
This particular biodiversity of peptaibols raises the question whether there is a correlation between peptaibols, which are often named after a putative producer species (harzianins, longibrachins, koningins, etc.), and the taxonomic status of the producer strains. The problem of the taxonomy of Trichoderma is difficult, and use of phenotypic species identification alone (as in most of these studies) is highly prone to error (Druzhinina et al., 2006). In addition, some peptaibols had already been described before 1969, a time when Trichoderma was believed to consist only of a single species, Trichoderma viride (Rifai, 1969), and many more up to 1991, when Trichoderma was believed to consist only of nine species aggregates (Bissett, 1984, 1991a–c, 1992). Consequently, the species identity of most of the strains producing known peptaibols up to that time is highly uncertain. Only recently have strains with defined species identity been investigated for the peptaibols they produce, which has led to the identification of several new compounds (Wiest et al., 2002; Degenkolb et al., 2006a, b; Vizcaino et al., 2006). In the latter study, peptaibols produced by five defined species of Trichoderma (T. pubescens, T. strigosum, T. spirale, T. erinaceum and T. stromaticum) were compared, and the authors concluded that the type of peptaibols produced correlates only poorly with the taxonomy of the species. This claim must be accepted with caution, however, as Degenkolb et al. (2006b) only investigated five out of more than 100 accepted species (cf. www.isth.info), and moreover included three species (T. pubescens, T. erinaceum and T. strigosum) which are phylogenetically closely related.
We have investigated peptaibol production by intact-cell MALDI-TOF mass spectrometry (ICMS) in 28 Trichoderma/Hypocrea species that represent a wide range of the diversity within this genus. ICMS has the advantages of being very rapid, using small samples (subcolony amounts) and requiring only minimal sample preparation. ICMS spectra in the range below 2 kDa identify characteristic metabolites, e.g. of bacilli (Williams et al., 2002; Pabel et al., 2003) and cyanobacteria (Erhard et al.,1997; Welker et al., 2002, 2004a, b), and we show here that highly characteristic peptaibol spectra can readily be obtained from microgram amounts of mycelia, thus providing characteristic metabolite fingerprints. A comparison of these fingerprints with molecular phylogenies of the species documents some correlation between types of peptaibol formed and phylogeny.
We have further classified the structures of 18- to 20-residue peptaibols and defined conserved residues corresponding to conserved domains of the respective non-ribosomal peptide synthetases (NRPSs). Sequences of conserved adenylate domains of synthetases forming trichovirins, paracelsins and atroviridins have been identified from strains representative of different sections of the genus. Substrate binding site analysis permits the prediction of substrate profiles and domain position. Analysis of the nucleotide sequences of the Aib-, Pro- and Gln-activating domains displays similar Kimura distances of the NRPS genes and housekeeping genes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Microbial strains and cultivation.
The Trichoderma/Hypocrea strains used in this study are listed in Table 1. All of them have been identified previously at the species level, and the corresponding references are given in Table 1. All strains were cultivated on malt extract agar (3 %) at 26 °C.
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Microbial characterization by MALDI-TOF MS analysis.
MS measurements were performed on a VOYAGER DE-PRO-TOF mass spectrometer from Applied Biosystems. Mass spectra were acquired in linear delayed extraction mode using an acceleration voltage of 20 kV and a low mass gate of 1500 Da. For desorption of the components, a nitrogen laser beam (
=337 nm) was focused on the template. The laser power was set to just above the threshold of ionization. Spectra for individual specimens were compiled, averaging results from at least 100 shots taken across the width of the specimen for m/z values of 2000–20 000.
Measurements were performed in delayed extraction mode, allowing the determination of monoisotopic mass values. A low mass gate of 800 Da improved the measurement by filtering out the most intensive matrix ions. The mass spectrometer was used in positive ion detection and reflector mode. In addition, an identification took place by a comparison between calculated and detected mass data.
In silico retrieval and identification of conserved adenylate domains.
A full-length copy of T. atroviride tex1 was identified in its genome sequence by a TBLASTN search with the T. virens tex1 gene as probe. The amino acids activated by individual A (amino-acid-activating)-domains were identified by a BLAST search with the corresponding domains of H. virens and H. jecorina (T. reesei; H. von Döhren & C. P. Kubicek, unpublished). The paracelsin synthetase gene has been identified in the H. jecorina genome sequence (T. reesei v2.0) and is partially annotated at http://genome.jgi-psf.org/cgi-bin/dispGeneModel?db=Trire2&id=123610; A-domains have been identified by correlation with known paracelsin structures (H. von Döhren & others, unpublished). Characteristic A-domains were identified by positional conservation and non-ribosomal code analysis (Rausch et al., 2005).
Molecular phylogenetic methods.
Sequences of rRNA spacer regions ITS1 and 2, and rpb2 were taken from the multilocus database (www.isth.info), and were aligned using Genedoc 2.6 (Nicholas & McClain, 1987) and visually corrected. Phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987) with 1000 bootstrap replicates, using the computer program MEGA 3.1 (Kumar et al., 2004).
For phylogeny of the peptaibol sequences, the amino acid sequences were first transcribed into a binary matrix in which the presence of a given amino acid at a given position was coded as 1 and its absence as 0. One and zero were subsequently replaced by the nucleotides T and A, and the matrix transcribed into a nucleotide alignment appropriate for analysis in MEGA 3.1 as above.
Tests for evolutionary mechanisms.
To test the fit of the sequences to the model of neutral evolution, the D test statistic proposed by Tajima & Nei (1984) was computed with the DnaSP program (Rozas et al., 2003). The extent of nucleotide divergence was estimated by using the uncorrected p distance (Nei & Kumar, 2000). The proportions of synonymous (pS) and non-synonymous (pN) differences per site were calculated by the modified Nei–Gojobori method implemented in DnaSP (Zhang et al., 1998).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, b). Peptaibol masses are predominantly in the 1000 to 2000 Da range. All peptaibols from the 32 Trichoderma/Hypocrea strains were detected as sodium [M+Na]+ or potassium [M+K]+ ions directly from microgram amounts of unfractionated cells. Peptaibols are easily released from fungal mycelia grown on plates. Although the sensitivity of detection has not been tested, peptaibols might be detected in single mycelial segments, as peptides have been detected in microcolonies and single filaments of cyanobacteria comprising about 100 bacterial cells (Fastner et al., 2001; Welker et al., 2004a, b, 2006).
Verification of the method by application to H. lixii
To document the quality of our method, a representative spectrum is shown for H. lixii (T. harzianum CBS 226.95; Fig. 1). Three sets of mass peaks were observed, corresponding to three peptaibols of SF1, SF4 and a third yet unknown subfamily.
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). An identical set of mass peaks has recently been reported for H. lixii CECT 2413 (Vizcaino et al., 2006), for which the species identity to the H. lixii anamorphic state has also been established (Hermosa et al., 2004). We have detected sets with identical masses in 16 other strains used in this study (see Table 2). As we only have molecular masses of these compounds, we cannot fully identify the respective peptaibols, however.
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(see below). The nature of these presumably 16-residue peptaibols remains to be shown. This group of peptides has not been detected in strain H. lixii CECT 2413 (E. Monte, personal communication).
Set 3 in Fig. 1, characterized by the most abundant mass peak at m/z 1800, is a representative of peptaibol group 19 (Table 2), with similar masses to the well known SF1 18-residue trichokindins and trichorzins of the MA type (containing Val in the terminal position, differing from Leu in the HA type, and Trp or Phe in the PA type), isolated from a putative H. lixii from soil in Nara, Japan (Iida et al., 1994) and strain M-922835 (Goulard et al., 1995), respectively. We thus speculate that these two isolates may indeed be H. lixii as well.
Two additional sets of peaks with very low intensity can be seen between sets 2 and 3, and preceding set 1; they may belong to the 10- and 17-residue compounds, possibly derived by proteolytic cleavage, but they have not been investigated further.
Peptaibol formation by different species of the genus Trichoderma
Having assessed that our method works well and provides data comparable to peptaibol production data by others, we then analysed the production profiles of 34 strains representing 28 species (Table 2): in total, we could identify 26 groups of peptaibols by mass data. The masses resemble H+, Na+ or K+ adducts of peptaibols, and all spectra have been compared for overlapping profiles. To identify known peptaibols on the basis of their mass data and to recognize possible new structures, we compared them with the monoisotopic masses of the relevant peptaibols from the Birkbeck database and complemented it with recently published data (Degenkolb et al., 2006a, b; Krause et al., 2006; Krause, 2006), thereby combining analogues of single isolates into groups. Thus all alamethicins and all paracelsins are included in groups 26 and 25, respectively, with other SF1 members.
As is shown in Table 2, some groups combine several sets of compounds with identical masses. Thus group 1 combines the trikoningins and trichogins, 11-residue peptaibols of the SF5 type, both with masses of 1052, 1066 and 1082 Da. These differ in mass from 11-residue trichovorins and trichorozins of the SF4 type, defined as group 6.
Members of the 20-residue trichosporins, paracelsins, saturnisporins, suzukacillins, trichocellins, trichokonins, longibrachins and trichoaureocins all share masses 1922, 1936, 1950 and 1964 Da, and are thus combined into our group 25. The rationale of combining peptaibiotics by molecular masses originates from a comparison of their sequence data. While there are a small number of positions with defined functional properties, such as Gln/Asn or Pro, the vast majority of positions have variable aliphatic side chains. These include Ala, with successive additions of methylenes to arrive at Aib, Val, Iva, Leu or Ile. Respective peptaibol synthetase modules may have either defined or less defined specificities, thus leading to sets of compounds differing in masses by 14 Da. A phylogenetic analysis of adenylate domains indeed indicates a clustering of all of these domains (results not shown). Non-conservative substitutions, like Ala–Ser transitions introducing a functional hydroxy group or Leu–Phe replacing an aliphatic side chain by an aromatic residue, then lead to differing mass deviations. Observed mass groups can be considered as diagnostic data to identify either new compounds with yet unknown masses, or mass ranges extending from known groups to more or less hydrophobic analogues.
Of the 26 sets of mass peaks identified, 12 correlate with mass sets of known compounds. Most of the remaining unknowns are found in single strains only, and only a few are widely distributed. Most strains produce two or more peptaibol groups. Twenty strains of 18 species produce 11-residue compounds of the trichovorin/trichorozin type (group 6), and nine species produce 18- and 20-residue peptaibols of the trichokindin/trichorzin type (group 19) as well as the paracelsin/alamethicin type (groups 25 and 26). Hence this survey reveals that the diversity of peptaibol structures in Trichoderma/Hypocrea is far from being recognized. Of the 26 mass sets, 14 cannot be correlated with any known peptaibols, and are likely to be as yet undescribed compounds.
Distribution of SF1-type compounds
SF1 contains 24 named compounds with their respective analogues (Chugh & Wallace, 2001), of which 21 originate from strains of the genus Trichoderma/Hypocrea. An additional SF1 group of compounds, the trichoaureocins, has recently been described by Degenkolb et al. (2006b). All of these peptaibols range in size from 18 to 20 amino acid residues.
18-residue peptaibols.
We found five groups characterized by 18-residue compounds, i.e. groups 18, 19, 20, 21 and 22 (Table 2).
Group 18 has been defined on the basis of 12 analogues of the trichovirin structural family, which are formed by T. viride NRRL 5243 (=H. lixii) in submerged cultivation (Fig. 2; Jaworski et al., 1999). We have detected similar masses in T. asperellum, H. virens (the former ex-type strain of T. flavofuscum, a species name which has been abandoned) and H. tawa. Masses of trichovirin analogues may overlap with masses of the trichorzin HA type, but the dominating analogues have lower masses due to their lower Aib content. Our mass data correlate with the results of Brückner et al. (1985) for T. asperellum (then called T. viride) NRRL 5242, in which a family of 13 trichotoxins was described.
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Gly-2 and Ser-2Ala-2 transitions, and Phe/Trp aromatic terminal amino alcohols (group 22, trichorzin PA type) replacing Leu/Val, leading to different masses, but with identical invariant residues displaying similar properties (Fig. 2). Group 20 contains an unknown mass set found only in T. viride (=T. viridescens) ATCC 28020.
Group 21 contains the recently described trichostromaticin-type peptaibols found in T. stromaticum BBA 70636/70638 (Degenkolb et al., 2006b). We found a similar mass set in the ex-type strain of T. pubescens DAOM 162162.
Finally, group 22 corresponds to the trichorzin PA type, where a change of the terminal amino acid to Trp leads to a mass increase of 87 Da. The PA type has been found in the putative strain ‘H. lixii’ M-902608, selected by Roquebert and colleagues for its antagonistic properties (Duval et al., 1997; Leclerc et al., 1998a, b). We detected corresponding masses in H. strictipilosa (ex-type strain of T. fasciculatum CBS 118.72, a species now abandoned) and H. hunua.
19-residue peptaibols.
Mass groups 23 and 24 correspond to the trichostrigocin and trikoningin types, respectively. Our data with the ex-type strain of T. strigosum are consistent with a detailed analysis of the same strain and strain T. cf. strigosum CBS 119777 (Degenkolb et al., 2006a) which produce trichostrigocins and tricholongins, respectively.
20-residue peptaibols.
This group of long peptaibols can be separated into two mass groups (25 and 26), depending on whether the position 2 amino acid is Ala (paracelsin type) or Pro (alamethicin type). Differentiation of these two groups by molecular ions is not trivial, as the 16 Da difference can be reduced to only two mass units when conservative Ala/Aib, Aib/Val or Aib/Iva exchanges take place, and terminal deaminations at Gln may further lead to an increase in a single mass unit. We found the paracelsin type as expected in T. reesei, and its close phylogenetic neighbours H. schweinitzii (=T. citrinoviride) and T. longibrachiatum, but also in members of other clades/sections, such as T. pubescens CBS 119776, H. pachybasioides and T. strictipile.
In H. jecorina, the 20-residue paracelsin SF1 type has been investigated in strain QM 9414 (=ATCC 26921=IMI 192656) (Brückner & Graf, 1983; Brückner et al., 1984), and additional paracelsins have been detected in a commercial preparation obtained from Fluka BioChemika (Pocsfalvi et al., 1997). Our investigation of H. jecorina QM 6a, the progenitor of the cellulase-overproducing mutant QM 9414, has confirmed the presence of paracelsins. In addition, we detected two compound sets of 18 and 19 residues, presumably derived by N-terminal cleavage and deletion of 1 amino acid residue. Since the genome sequence database of H. jecorina QM 9414 (http://gsphere.lanl.gov/trire1/trire1.home.html) contains only two putative NRPS genes encoding 20- and 14-residue peptaibol synthetases, respectively (H. von Döhren & others, unpublished), these two sets of peptaibols originate as two products from one synthetase and one additional product by peptidolytic modification.
SF1 compounds of 20 (longibrachin) and 19 residues (tricholongins) have been described in a putative T. longibrachiatum isolate (Leclerc et al., 2001; Rebuffat et al., 1991; Auvin-Guette et al., 1992). We detected masses corresponding to longibrachins in the T. longibrachiatum ex-type strain CBS 816.68. T. longibrachiatum and T. ghanense, another member of section Longibrachiatum, have been shown to form eight longibrachin analogues, and various C-terminal degradation products have been investigated in detail by Krause (2006). Masses of the alamethicin type (group 26) have been detected in H. atroviridis P1=ATCC 74058, T. brevicompactum and T. hamatum.
Distribution of SF4 type of peptaibols
SF4 contains 11- and 14-residue compounds. The latter, best known as harzianins, have a characteristic spacing of three Pro residues in positions 5, 9 and 13. Interestingly, the 11-residue group can be derived from harzianins by internal deletions of the amino acids Leu, Aib and Pro. Work on H. lixii (Augeven-Bour et al., 1997) and H. jecorina (H. von Döhren & others, unpublished) suggests that both types of compounds originate from one peptide synthetase. Known compound structures have been compiled in Fig. 3.
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) correspond to 11-residue peptaibols; however, only group 6 compounds are known. These structures contain 5 invariable and 6 variable amino acid residues (Fig. 3). In addition to the two H. lixii strains mentioned above, the group 6 mass set has been found in 19 of the strains investigated (Table 1), and is thus the most widely distributed type of peptaibol. For descriptive purposes, members of SF4 group 6 can be characterized as 11-residue peptaibols of the type AcX(N,Q)XXUPXXUPXol, with fixed positions 5 (Aib), 6 (Pro), 9 (Aib) and 10 (Pro), Asn or Gln in position 2, X being variable amino acids, and a terminal leucinol or valinol. The monoisotopic molecular masses are 1161, 1175, 1189 and 1203 Da.
14-residue peptaibols.
Of the 14-residue type, 12 harzianins have been described with the general structure AcU(N,Q)LUP(A,S)(V,I)UP(J,U)LUP(L,V)ol, all isolated from the undefined H. lixii strain M-903603 (Rebuffat et al., 1995; Goulard et al., 1995). The similar trichovirin I type with the general structure Ac(U,J)(N,Q)LUP(A,S)VUP(J,U)LUPLol has been described in seven variants from an undefined strain of T. viride (Brückner & Koza, 2003). Note the conserved residues in the overall structure AcxxLUPxxUPxLUPxol, with all other residues undergoing conservative replacements with the exception of residue 6, which can be Ala or Ser. All 19 variants provide a set of eight monoisotopic masses of 1385, 1399, 1401, 1413, 1415, 1427, 1429 and 1443 Da. Respective MH+, MNa+ or MK+ ions were observed in groups 8, 9 and 11, however, with additional unknown masses. As the peaks of highest intensity range from m/z 1403 to 1468 and 1482 to 1496 (Table 2), these three groups have been defined accordingly. Groups 9 and 12 are in the same mass range, but do not correspond to known compounds. The low mass SF4 type has been detected in T. croceum and T. hamatum, an intermediate type in H. virens, and the high mass type in eight strains, including T. longipile, T. tomentosum, T. oblongisporum and H. semiorbis, all belonging to section Pachybasium B. We did not detect the SF4 type in our H. lixii and T. viride strains.
Distribution of SF5 type of peptaibols
The SF5 type comprises several lipopeptaibols. A group of octanoyl-capped 11-residue peptaibols, first described as trichogin/trikoningin from T. longibrachiatum and T. koningii (Auvin-Guette et al., 1992, 1993), has recently been extended by 14 lipostrigocins isolated from strains of T. strigosum and T. pubescens (Degenkolb et al., 2006b). In addition, 7- and 10-residue lipopeptaibols (lipopubescins) have been described, which are likely to originate from a closely related peptaibol synthetase, or even a single synthetase. Also, trichodecenins, 7-residue decenoyl-capped peptaibols, isolated from an uncharacterized T. viride (Fujita et al., 1994), are structurally related.
All 11-residue compounds have the structure Oc(U,Vx)G(Vx,Lx)UGG(Vx,Lx)UG(I,Lx)Lol, where Oc=octanoyl, Vx=Val/Iva and Lx=Leu/Ile, with possible monoisotopic masses of their Na+ adducts of 1061, 1075, 1089, 1103 Da. This group has indeed been detected as expected in T. koningii, T. strigosum, T. pubescens, and also in T. hamatum, which are all members of the section Trichoderma. We have not detected this group in T. longibrachiatum from section Longibrachiatum.
Phylogenetic analysis of peptaibol formation and correlation with species phylogeny
Having assessed the peptaibol pattern formed by the 28 species of Trichoderma, we investigated whether the formation of any of the groups would correlate with individual phylogenetic clusters or branches. To this end, we used a fragment of the RNA polymerase subunit B gene, which has previously been shown to result in the most reliable and resolved phylogeny of all marker genes available today (Druzhinina et al., 2005; Druzhinina & Kubicek, 2005). The results (Fig. 4) confirm the established clusters for sections Longibrachiatum, Trichoderma and the Pachybasium B clade, as described by Kullnig-Gradinger et al. (2002). Correlation of the individual groups of peptaibols with these clusters appears to be poor.
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One might argue that the fine structure (=sequence) of peptaibols within the same group might still be different enough to attribute individual compounds to selected phylogenetic clades. We tested this with group 25 and 26, which both include 20-residue peptaibols (section Longibrachiatum – longibrachins, trichobrachins, trichoaureocins, paracelsins and saturnisporins; section Trichoderma – atroviridins; section Pachybasium B – polysporins and alamethicins). To this end, the peptaibol amino acid sequence was transformed into a binary matrix, which also took the ambiguity of individual positions into account, and then this matrix was transformed into an artificial nucleotide sequence on which we performed neighbour-joining analysis. The results show that this approach grouped the 20-residue peptaibols into five clusters whose members shared the highest similarity (Fig. 5). These clades exhibited no relationship to gene phylogeny, however. We conclude that the biodiversity of peptaibols of Trichoderma is not concordant with the evolution of the genus, but still contains a background of ancient species-specific patterns.
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). In the meantime, the number of known peptaibol sequences has more than doubled. Relevant new structures have been included in the Discussion. In the genus Trichoderma/Hypocrea, so far only SF1, 4, 5 and 9 have been described (Szekeres et al., 2005).
SF1 comprises more than half of all the structures which are characterized by chain lengths between 18 and 20 residues. These ‘long’ peptaibols all share a Gln residue in position 6 or 7 which fundamentally contributes to the channel-forming properties (Chugh et al., 2002). In addition, one or two Gln/Glu residues are consistently located in the C-terminal region, preceding the terminal alcohol.
To further classify members of SF1, various approaches are possible. We have started from plain chemical systematics, which defines types of 18, 19 or 20 residues, and have then further compared these types with respect to the positions of functional side chains and sequence similarities. By using this approach, we can define nine major types, as shown in Fig. 6, within this family. Types I and II are all 18-residue peptaibols with Gln in position 6 and either Gly or Ser/Ala in position 2. It is also intriguing that all of them contain Aib in position 1, and the sequence Pro-Leu-Aib in positions 13–15. Type III peptaibols are 18-residue compounds with Gln in position 7. It is noted that in this case Ala is conserved in position 2, and there is an amino acid insertion at position 6 with simultaneous loss of 1 amino acid between residues 9 and 12. Type IV peptaibols are 18-residue peptaibols, similar to type III, but with an additional Gln in position 11. Among the 19-residue peptaibol types (V–VII), type V is characterized by a Phe inserted in position 1, thus moving Gln at position 6 to position 7. Type VI mostly displays a Phe in position 3, but this position is sometimes substituted by Aib. Types VIII and IX are all 20-residue compounds with either Pro or Ala/Gly in position 2, respectively, and which also show conservation of Aib-1, Gln-7, Pro-14, Aib-16, Gln-18 and Gln-19. It is intriguing to note that type VII peptaibols may have been derived by deletion of residue 6 from type VIII (trichokonin V). However, peptaibols of this type are also found as a unique set of compounds (e.g. trichorzianins and trikoningin).
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) is compared with their established phylogeny, some concordance is observed: e.g. species from section Longibrachiatum produce both type VIII and type IX compounds, but none of the shorter types I–VI. On the other hand, strains of the section Trichoderma B contain the longer types VIII and IX, the short types I–III and type IV, which is not produced by strains from other sections. In addition, H. pachybasioides has been reported to produce type VIII and IX peptaibols (Iida et al., 1990, 1993, 1999; New et al., 1996; Fujita et al., 1981), but the actual strain has not yet been identified by sequence analysis. Notably, only one type of SF1 peptaibol is found in each strain, with the exception of type VII which probably arose by a biosynthetic deletion of the position 6 residue from a corresponding type VIII peptaibol.
Conserved features within SF1 type peptaibol synthetases
In view of the fact that peptaibol synthetases belong to the NRPSs, they are expected to contain, in addition to the unique N-terminal acetyltransferase domain and the C-terminal reduction domain, 18–20 modules of a sequence of an amino-acid-activating adenylation domain, a carrier protein domain and a condensation domain. Each of the positional boxes in Fig. 6 in theory therefore predicts the organization of modules in the respective NRPSs. So far, the gene encoding trichovirin synthetase from H. virens (section Pachybasium B) has been isolated and the amino acid sequence of the respective NRPS has been deduced. In addition, putative peptaibol synthetase genes have been detected in the genome sequences of T. reesei (section Longibrachiatum) and H. atroviridis (section Trichoderma) (H. von Döhren & others, unpublished). These three synthetases produce ‘long’ peptaibols of SF1 type I (trichovirins), SF1 type VIII (paracelsins) and SF1 type IX (atroviridins). In addition, the terminal region of an SF1 type I peptaibol synthetase from H. lixii (section Pachybasium B) has been sequenced (Vizcaino et al., 2005). No genes encoding peptaibol synthetases synthesizing products of SF4, 5 and 9 have been cloned yet.
Phylogenetic analysis of conserved adenylation domains of SF1 type synthetases
Since the peptaibols produced by these strains differ in some of their modules, the use of the full-length amino acid sequence for phylogenetic analysis is not possible. We therefore decided to only use positionally conserved domains in the respective synthetases. As shown in Fig. 6, such domains are located in the acyl transferase modules, in the Aib-adding module 2 (with the exception of type V found in boletusin, peptaivirins and chrysospermins produced by strains whose species identity is uncertain), in the positionally conserved central Gln module (position 6 or 7), in the Pro modules (positions 13 or 14), in the Aib module (positions 15 or 16) and in the late Gln modules (positions 18 and 19), although the origin of these tandem Gln modules in types VIII and IX may be due to module duplication and their use in phylogenetic analysis would thus be corrupted. We therefore compared nine Gln-activating domains from trichovirin synthetase, paracelsin synthetase, atroviridin synthetase and a type I NRPS from H. lixii, as well as early Aib-activating domains from the first NRPS modules, Pro-activating domains and late Aib-activating domains following next-but-one to the Pro domains. The phylogenetic analysis shows that the domains cluster with high bootstrap support not only with respect to the recognized amino acid, but also with respect to their relative position within the peptaibols (Fig. 7a). Similar phylogenetic patterns were obtained when only the Gln-activating domains or only the Pro- and Aib-activating domains were analysed (data not shown). When the branching pattern within these clusters was compared to the established species phylogeny based on rpb2 or ITS1 and 2, discordance was noted (Fig. 7b). Within most clusters, the domains from H. virens and H. jecorina were most similar, and H. lixii was most distant. These data indicate that the phylogeny of the adenylation domains for these amino acids does not parallel the evolution of these three species.
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) with the phylogenetic separation of early or central Gln domains, late Gln domains and late additional Gln domains (Table 5) enables refinement of the substrate-binding pocket consensus.
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and Challis et al. (2000), of early and late domains differ by a single TyrPhe replacement; however, in the extended binding site analysis 3 of the 34 amino acids are different. Extended binding site analysis of the Pro adenylation domain reveals the complete identity of all 34 residues. Phylogenetic and comparative sequence analysis thus permits not only a prediction of the substrate profile, but also the classification of the domain position within the synthetase.
Nucleotide sequence evolution of substrate-activating domains
To learn more about the evolution of the peptaibol synthases, we have investigated their interspecies nucleotide diversity, rate of nucleotide substitution and nucleotide sequence phylogeny. The latter provided the same phylogenetic tree structure as the amino acid sequences (data not shown). Applying Tajima's test implemented in the program DnaSP confirmed that they are undergoing neutral evolution (D=2.65008 and 1.874744 for the Aib/Pro- and Gln-activating domains, respectively), which coincides with a high KS/KN (differences in synonymous to non-synonymous substitutions) ratio (>5) which indicates the operation of purifying selection. Nucleotide diversity (p) was between 0.34 and 0.36, which correlates well with a similar value for nucleotide diversity in the exons of tef1 and rpb2 (0.33 and 0.32, respectively). In summary, the activating domains for Aib, Pro and Gln have evolved in a similar neutral way as housekeeping genes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Krause et al., 2006), our approach only partially identifies the peptaibols, but the information can be obtained from extremely small amounts of biomass and much more rapidly. Below, we discuss the consistency of our data with the available structural data and the implications of molecular genetic data for the study of the evolution of these NRPS systems.
Peptaibol production data survey and correlation of groups defined in our survey with known compounds
We have compiled available data on peptaibols described for Trichoderma/Hypocrea (Table 3 and 4), including some mass data permitting comparison to Table 2. Unfortunately, as stressed in the Introduction, the species identity of several strains in the Birkbeck database is highly doubtful, and we present these data and additional data therefore in two separate tables; species identities quoted in Table 4 must be used with caution. Having defined 26 peptaibol groups by their mass profiles, we first need to ask if our data correlate with available data and if additional compounds are known from strains outside our selection.
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investigated 11 defined strains of Trichoderma/Hypocrea species by solid phase extraction/liquid chromatography/electrospray ionization/mass spectrometry (SPE/LC-ES-MS) (T. asperellum CBS 433.97, T. inhamatum CBS 345.96=H. lixii, T. aggressivum f. europaeum CBS 100526, T. stromaticum CBS 101875, H. semiorbis CBS 244.63, H. vinosa CBS 247.63, H. dichromospora CBS 337.69, H. gelatinosa CBS 724.87, H. nigricans MUCL 28439=H. lixii, H. muroiana MUCL 28442 and H. lactea CBS 853.70). Molecular masses and partial sequences of 48 peptaibols have been compiled, only seven of which match data compiled in the Birkbeck database, the remaining being either new analogues or new types of compounds. Interestingly these 48 compounds can be classified into six defined groups as shown in Table 1 and six new groups not yet defined. As only four of the above strains were contained in our pool, the results of Krause et al. (2006) can be used to amend our data and extend the number of mass-defined groups to 32 within a total of 39 strains.
Mass peaks may be composed of several peptaibols.
In the case of H. gelatinosa, this study defined compounds with masses of 1176 and 1204 Da with partial sequences Vxx(=Val/Iva)-Lxx(=Leu/Ile)-Lxx-Aib-Vxx and Gln-Lxx-Lxx-Aib, respectively. The Na+ adduct of the 1204 Da compound would correlate with m/z 1227 and the sequence data of group 6. The partial sequence and mass data of the 1176 Da compound are unknown. As the respective Na+ adduct would also fit into group 6, these data warn us that unknown compounds may be hidden in the known masses. In addition to these two 11-residue peptaibols, we also detected group 11 and group 19 peptaibols. In the case of H. semiorbis, Krause et al. (2006) described 20-residue compounds with masses of 1937, 1951 and 1965 Da, corresponding to group 25 compounds as respective Na+ adducts with m/z 1960, 1974 and 1988. Surprisingly, we detected groups 6, 11, 13 and 19. Here, apparently, strain identity needs to be confirmed.
Strains of one species may produce different peptaibols.
Krause et al. (2006) found that H. lixii CBS 273.78 produced three sets of compounds with masses of 1146, 1675 and 1703/1717/1731 Da. The long peptaibols clearly match group 18, the trichovorin II type, while the others have no match, presumably representing new groups. This agrees with H. lixii CBS 226.95 which produces 18-residue trichotoxins, also members of this mass group. In our study, H. lixii CBS 273.78 produced compounds of groups 6, 14 and 19. These data indicate that strains genetically identified as H. lixii may still differ in their peptaibol production profiles. However, H. lixii is a complex of several cryptic species (Chaverri et al., 2003) which are known to differ in their biochemical and physiological properties (C. P. Kubicek & I. S. Druzhinina, unpublished data).
Putative bona fide H. lixii strains (Table 4), trichorzins of the HA (group 18) and MA types (group 19) have been described from strains M-903602 (soil sample, Uruguay) and M-922835 (soil sample, Malaysia) (Goulard et al., 1995; Hlimi et al., 1995). A set of trichotoxins (group 18) with 11 of the 22 analogues already known from H. lixii NRRL 5243 (Przybylski et al., 1984; Brückner et al., 1984, 1985) have been described from Trichoderma sp. strain PC01 (Suwan et al., 2000). In addition, harzianins with 11 and 14 residues (groups 6 and 11) and trichorozins (group 6) have been described from other putative strains of H. lixii. These data are consistent with the conclusion that H. lixii produces either group 18 or 19 compounds, but neither group is restricted to this species. The transition from group 18 to 19 involves a substrate specificity shift of domain 2 from Gly to Ser. Such a shift might occur by point mutation or domain exchange. The unique group 22, corresponding to harzianins of the PA type with a terminal aromatic residue, described for the undefined strain M-902608, has also been detected in T. hunua and T. strictipilosa, both members of section Pachybasium B. These data may indicate that the occurrence of 18-residue peptaibols terminating in an aromatic amino alcohol may be restricted to this section.
A similar conclusion can be reached considering the recent study of Degenkolb et al. (2006b) comparing various strains of T. brevicompactum (Table 3). All four strains produced alamethicins, trichocryptins A and trichobrachins, while only two strains produced trichocryptins B in addition. Comparing three strains of T. cf. brevicompactum, the same study describes trichocompactins and trichobrevins in CBS 119576, alamethicins and trichocryptins in CBS 119577, and alamethicins, trichoferins, trichocompactins and trichobrevins A and B in NRRL 3199.
In a recent peptaibiomics study, plant-protective Trichoderma strains of different origins (T. strigosum, T. pubescens and T. stromaticum) were compared (Degenkolb et al., 2006a). The authors concluded from the differences between the strains and similarities between different species that peptaibol sequences are of limited relevance for chemotaxonomy. Thus T. strigosum CBS 119777, a German compost isolate, was found to produce tricholongins B, 19-residue peptaibols of type VI/SF1, while T. strigosum CBS 348.93, a forest soil isolate from North Carolina, produced trichostrigocins, 19-residue type VII/SF1 compounds. There are three significant module changes in these types, exchanging Gly with Ala, Phe with Aib and Aib with Lxx (Fig. 6). Interestingly, a tricholongin has also been detected in T. cf. pubescens BBA 66989. The 11-residue lipostrigocins were detected in all three strains. Both species belong to the section Trichoderma. Other type VII/SF1 compounds, the 19-residue trichorzianins and trikoningins, are produced by H. atroviridis ATCC 36042 and T. koningii, respectively, also members of this section.
Another example of significant alteration of a structure is the production of lipostrigocins B and linopubescins in T. cf. pubescens BBA 66989 and T. pubescens CBS 345.93, respectively (Degenkolb et al., 2006a). Interconversion of the 11- and 10-residue compounds would require alteration (Gly/Vxx) and deletion/insertion of one module, probably involving extensive alterations of the respective NRPS gene.
From the limited data available, it thus seems possible that certain structural types are restricted to sections of the Trichoderma/Hypocrea group. It will be interesting to investigate whether the occurrence of similar compounds in different species is due to coevolution or to horizontal gene transfer.
Failure to detect certain compounds.
The unique trichostromaticin type of 18-residue peptaibols (group 21, type III of SF1) has been found in T. stromaticum BBA 70636/70638 (Degenkolb et al., 2006a). In this case we failed to detect peptaibols, although similar growth conditions were used. In T. stromaticum CBS 101875, in addition to group 21 trichostromatocins C, group 13 peptaibols have been described (Krause et al., 2006). The unknown 1538 Da compound detected might be identical to compounds we detected in H. atroviridis ATCC 74058, H. citrina, T. hamatum, T. longipile, T. pubescens, H. semiorbis and H. strictipilosa.
In a putative strain of T. pubescens (BBA 66989=DAOM 162162), Degenkolb et al. (2006b) found a 19-residue compound of the tricholongin type and group 1 compounds of the lipostrigocin type. We detected only group 1 and 13 mass peaks in this strain, and failed to find the long peptaibol. T. pubescens CBS 119776, however, was found to produce a 20-residue peptaibol of the group 25 type. Thus, as in H. lixii, strains of T. pubescens produce different groups of 19- and 20-residue peptaibols.
Failures in compound detection can be related to either strain-specific production profiles of further isolates, whose species identity has been verified by sequence analysis, or loss of production by gene loss or expression failure. Both cases have been well documented in cyanobacteria, where numerous isolates from single biotopes showed a large variety of chemotypes. As in the case of Trichoderma/Hypocrea, the compounds derived from NRPS genes in cyanobacteria have been used for chemotype definition (Welker et al., 2004a, b, 2006).
Possible identities of undefined strains.
Mass group 24 corresponds to the 19-residue trikoningin type, and trikoningin KA has been found in a putative T. koningii isolate from Uruguay (Auvin-Guette et al., 1993). We detected masses corresponding to trikoningin in T. koningii CBS 460.96. We did not see, however, the 20-residue trichokonins, belonging to mass group 25, which have been described from another putative T. koningii isolate (Huang et al. 1995a, b, 1996; Landreau, 2001; Landreau et al., 2002; Table 3). T. koningii has frequently been confused with Trichoderma species from section Longibrachiatum (Lieckfeldt et al., 1999).
Trichorzianins (SF1 type VII), which are 19-residue peptaibols with 13 analogues in the biocontrol strain T. atroviride P1 (described as H. lixii in this study; Pocsfalvi et al., 1998), of which eight have already been described (Bodo et al., 1985; Rebuffat et al., 1989), and the 19-residue peptaivirins (SF1, type V) from an unidentified Trichoderma species (Yun et al., 2000) have not been included in this study, but would need to be described in two additional mass groups (Table 4). In this study we failed to detect these compounds in strain P1. Instead, we found 20-residue peptaibols of group 26 corresponding to the alamathicin type, including atroviridins, well known from strains H. atroviridis P1 and ATCC 36042 (Oh et al., 2000). Unless the strain has been confused in the earlier study, these differences must be due to different culture conditions.
The structures of atroviridins and trichorzianins deviate in various positions, so that a direct transition by gain or deletion of one module is not possible. Interestingly, strain H. atroviridis IFO 31288, formerly described as H. muroiana, produces the 18-residue hypomurocins (Becker et al., 1997), members of group 19, which cannot be distinguished from other members of this group, trichokindins and trichorzins type MA, by molecular ions alone. Thus all three characterized strains of H. atroviridis produce different types of SF1 peptaibols, 18-residue hypomurocins (type II, group 19), 19-residue trichorzianins (type VII, no group assigned) and 20-residue atroviridins (type IX, group 26). Other group 26 producers are strains of T. brevicompactum, which have been extensively studied by Degenkolb et al. (2006b) and shown to produce alamethicins of the F-30 type. Contrary to H. atroviridis, section Trichoderma, these belong to section Pachybasium B. There are only minor differences between atroviridins and alamethicins: the specificity of position 17 is shifted from Aib to Iva/Aib, and in the case of alamethicin, Glu-18 may replace Gln-18.
Twenty-residue compounds of the paracelsin type (group 25, type VIII) are the only long peptaibols found in section Longibrachiatum. We have also found masses corresponding to group 25 in section Pachybasium strains H. pachybasioides DAOM 167068 (formerly T. croceum) and H. strictipilosa CBS 347.93. Group 25 compounds have also been described for two other putative strains of H. pachybasioides, i.e. trichosporins (Iida et al., 1990, 1993, 1999; Sharman et al., 1996) and polysporins (type VIII) (New et al., 1996; Fujita et al., 1981).
These data imply that group 25 peptaibols are the only long types found in section Longibrachiatum, while strains in section Pachybasium produce a variety of long types, including this group. Group 26 on the other hand has so far only been found in sections Trichoderma and occasionally in Pachybasium B.
More than one peptaibol produced from a single synthetase? The case of SF4 11- and 14-residue compounds.
Both 11-residue and 14-residue peptaibols have been assigned to SF4 as their structural similarity implies some relationship which has not yet been defined. Group 6 compounds with the structure AcX(N,Q)XXUPXXUPXOH (Fig. 3) and groups 8, 9 and 11 with the general structure AcX(N,Q)LUPXXUPXLUPXOH have identical terminal regions (underlined) and share six of the central residues (XXUPXX). Biosynthetically, a deletion of modules 3–5 (LUP) of a putative 14-module NRPS would lead to group 6 compounds. Two 11-residue harzianins, HB I and HK VI, have been isolated from H. lixii M-903603 (Augeven-Bour et al., 1997) and T. pseudokoningii MVHC 662 (Rebuffat et al., 1996), respectively. While HB I represents 14-residue SF4 peptaibols of the same strain with an internal deletion of 3 residues, HK is a member of a prominent family of 11-residue peptaibols which have not been structurally characterized. Both structures could be derived from the same deletion. As both 11- and 14-residue compounds have been detected in one strain, M-903603, a single NRPS might be suspected. It is not known, however, if the T. pseudokoningii strain also produces 14-residue compounds.
We have detected the formation of both 11- and 14-residue compounds of groups 6 and 9, 10 or 11 in several strains of section Pachybasium B (Table 2). However, group 6 compounds are widely distributed in other sections as well. Indeed, a more detailed analysis of H. jecorina (section Longibrachiatum) showed the presence of a 14-module NRPS, although some strains were found to produce only 11-residue group 6 compounds, while other strains produced both 11- and 14-residue peptaibols (H. von Döhren & others, unpublished). It remains to be shown if these NRPSs show section- or strain-specific alterations.
Phylogenetic analysis of peptaibol synthetases hints at a complex history.
A comparison of sequence data for conserved amino-acid-activating domains of long SF1 peptaibols reveals a similar phylogenetic distance in the peptaibol synthetase genes to that in other housekeeping genes, indicating a comparable rate of evolution. Genetic data from NRPS systems within the Trichoderma/Hypocrea group may thus reflect, at least within the conserved regions, the distances obtained from the comparison of other genes.
These findings only partially agree with recent work on cyanobacterial NRPS systems. The characteristic conserved regions of the NRPS genes of Cyanobacterium species synthesizing cyclic 7-residue microcystins, and which are produced by different sections of the cyanobacteria, have been compared using sequences from both 16S rRNA and a fragment of the RNA polymerase I gene (Rantala et al., 2004). Phylogenetic analysis indicates concordant evolution of these housekeeping genes with the NRPS genes for their entire evolutionary history, and does not support horizontal transfer between the genera. Our study indicates that a similar form of evolution has also taken place in the peptaibol synthetase genes.
A more complex view of peptaibol chemotypes.
The sporadic distribution of bacterial peptide and toxin gene clusters has been ascribed to repeated losses during diversification processes. In the case of peptaibols, we also see a patchwork-like distribution of products, and even strains of the same (confirmed) species may display variable product patterns. Such distributions have been analysed by employing MALDI-TOF MS typing of single filaments or microcolonies of cyanobacteria characterizing single clones within defined biotopes (Fastner et al., 2001; Welker et al., 2002, 2004a, b, 2006; Welker & von Döhren, 2006). In one example, 18 clones isolated from single filaments of Planktothrix from a single lake represented 15 chemotypes with respect to at least 11 different non-ribosomal peptides, including 25 analogues. The number of peptides produced has been found to range from one to four, indicating a limited coding space within the bacterial genome. Likewise, our data indicate a variety of chemotypes of individual species, but the number of peptaibols produced seems to be restricted to at most four types.
In bacteria, these observations have been interpreted as an exchange of NRPS gene clusters between strains of the same species, since transposase-related sequences have frequently been found at cluster boundaries. Comparing the genomes of Bacillus subtilis 168 and the closely related Bacillus amyloliquefaciens FZB 42, three and six NRPS/PKS clusters were found, respectively (Koumoutsi et al., 2004). Two NRPS clusters and operons were positionally conserved with respect to B. subtilis, and several transposase sequences are contained in it. As for fungi, transposon relics have so far only been found in the ergovaline biosynthetic clusters of the endophyte Epichloe (Fleetwood et al., 2007). Interestingly in Trichoderma, similar genes are found 5' and 3' of the atroviridine synthetase of H. atroviridis and of a putative harzianine synthetase in H. jecorina which encode SF1 and SF4 peptaibol types, respectively (H. von Döhren, unpublished data). These findings indicate that transfers and exchanges of peptaibol synthetase genes may also contribute to the chemodiversity of peptaibols in the Trichoderma/Hypocrea group, and account for the diverse chemotypes of single species. However, the diversity of the peptaibols formed also appears to be related to certain pools of compounds restricted to sections or clades. It is possible that the formation of certain chemotypes could be traced back to gene transfer events between closely related species. We must admit, however, that this has been shown here only for a small number of genes, and exceptions caused by horizontal recombination events or recombination events between synthetases of different subfamilies cannot be fully ruled out at this stage.
Conclusions
Many peptaibiotics have been named after the Trichoderma species producing them. Unfortunately, the identity of most of these Trichoderma strains must be treated with extreme caution, due to the difficulties of identifying these species by morphological means only (cf. Druzhinina et al., 2006). Krause et al. (2006), investigating five defined, different species of Trichoderma, recently showed that even closely related species can produce significantly different peptaibol patterns. Two studies comparing in detail two or more different strains from various species even demonstrated strain-specific production of certain peptaibols (Degenkolb et al., 2006a, b). The present study, which made use of a set of 28 phylogenetically characterized species, basically supports these findings. However, a more detailed inspection shows that some generalizations are still possible, considering in more detail the structural types of peptaibols and conserved structures of peptaibol synthetases. Some types of compounds defined by mass groups or sequence-based types seem to be restricted to single sections or clades. The validity of these predictions can be documented by the findings that a strain of H. peltata has been reported to produce group 25 peptaibols, which we interpret to be typical for section Longibrachiatum. Although an rpb2 sequence for H. peltata was unavailable for this study, ITS1 and 2 sequence analysis places it in a sister clade close to section Longibrachiatum (Dodd et al., 2002; Druzhinina et al., 2005) which is consistent with this prediction. In addition sequence data of the respective peptaibol synthetase genes may provide information on their phylogenetic background.
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ACKNOWLEDGEMENTS |
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Edited by: N. L. Glass
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 5 February 2007;
revised 14 May 2007;
accepted 22 June 2007.
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