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Sylvie Rebuffat - One of the best experts on this subject based on the ideXlab platform.
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Identification of Peptaibols from Trichoderma virens and cloning of a Peptaibol synthetase
The Journal of biological chemistry, 2002Co-Authors: Aric Wiest, Sylvie Rebuffat, Christophe Goulard, Bernard Bodo, Darlene Grzegorski, Daniel J. Ebbole, Charles M. KenerleyAbstract:The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high alpha-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a Peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virens was shown to produce a mixture of Peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all Peptaibol isoforms. Identification of the gene responsible for Peptaibol production will facilitate studies of the structure and function of Peptaibol antibiotics and their contribution to biocontrol activity.
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sequences and antimycoplasmic properties of longibrachins lgb ii and lgb iii two novel 20 residue Peptaibols from trichoderma longibrachiatum
Journal of Natural Products, 2001Co-Authors: G Leclerc, Christophe Goulard, Yann Prigent, Bernard Bodo, Henri Wroblewski, Sylvie RebuffatAbstract:Longibrachins are members of the class of natural Aib-containing peptides designated as Peptaibols. Six longibrachins, LGA I−IV and LGB II and III, were purified from a Trichoderma longibrachiatum strain by a procedure employing several chromatography steps including reversed-phase HPLC. The amino acid sequence determination was based on a combination of liquid secondary ion mass spectrometry (LSIMS) and two-dimensional 1H and 13C NMR spectroscopy. Longibrachins are 20-residue Peptaibols with a C-terminal phenylalaninol and either neutral (LGA; Gln18) or acidic (LGB; Glu18) character. Longibrachins LGB II and III have novel sequences. Both longibrachins LGA and LGB show significant bactericidal activity against mycoplasmas (Acholeplasma, Mycoplasma, and Spiroplasma), with minimal inhibitory concentrations in the range 1.56−12.5 μM (3−25 μg/mL), and also perturb the permeability of membrane bilayers. Longibrachin LGA IV is the most potent of the presently known 18−20-residue Peptaibols. The antimicrobial a...
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two unprecedented natural aib peptides with the xaa yaa aib pro motif and an unusual c terminus structures membrane modifying and antibacterial properties of pseudokonins kl iii and kl vi from the fungus trichoderma pseudokoningii
Journal of Peptide Science, 2000Co-Authors: Sylvie Rebuffat, Christophe Goulard, Sanae Hlimi, Bernard BodoAbstract:Pseudokonins KL III and KL VI are two natural ten-residue peptides, which both contain the (Xaa-Yaa-Aib-Pro) motif and exhibit an unusual C-terminus. They have been isolated from the fungus Trichoderma pseudokoningii by intensive reversed-phase HPLC, beside Peptaibols classically C-ended by a beta-amino alcohol. The amino acid sequences and the chemical structures of the C-ends have been determined by the combined use of positive ion LSI-MS and two-dimensional homo- and heteronuclear NMR, including COSY, TOCSY, ROESY, 13C heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC). Instead of one of the amino alcohols usually found as C-terminal residue in Peptaibols, pseudokonins KL III and KL VI are characterized by -Pro-NH2 and cyclo-(Aib-L-Proal) (Proal, prolinal), respectively. Such backbone modifications are described here for the first time for Peptaibol antibiotics. The unusual cyclo-(Aib-L-Proal) C-terminus is probably the result of an intramolecular cyclization of the two last Aib and Pro residues of a ten-amino acid precursor, via a Proal intermediate. A secondary structure stabilized by -C=O...H-N-hydrogen bonds of the 1<--4 type has been deduced for both peptides from ROESY data, 3JNHCalphaH couplings and amide proton temperature coefficient values. The (Xaa-Yaa-Aib-Pro) beta-bend ribbon spiral, which has been described for the first time in the case of a 14-residue Peptaibol containing three repetitive (Xaa-Yaa-Aib-Pro) motifs (Segalas G et al. Biopolymers 1999; 50: 71-85) appears to be maintained in the two shortened modified peptides. The beta-bend ribbon structure thus appears to be initiated by a single (Xaa-Yaa-Aib-Pro) motif and unaffected by the C-terminal modifications. However, the membrane and antibiotic properties of pseudokonins KL III and KL VI, point to the unfavourable effect of both shortening and cyclization of the peptide chain.
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characterization of a type of bend ribbon spiral generated by the repeating xaa yaa aib pro motif the solution structure of harzianin hc ix a 14 residue Peptaibol forming voltage dependent ion channels
Biopolymers, 1999Co-Authors: Isabelle Segalas, Bernard Bodo, Yann Prigent, Daniel Davoust, Sylvie RebuffatAbstract:The three-dimensional solution structure of harzianin HC IX, a Peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two-dimensional nmr spectroscopy combined with molecular modeling. This 14-residue peptide, Ac Aib1 Asn2 Leu3 Aib4 Pro5 Ala6 Ile7 Aib8 Pro9 Iva10 Leu11 Aib12 Pro13 Leuol14 (Aib, α-aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short-sequence Peptaibol class characterized by an acetylated N-terminus, a C-terminal amino alcohol, and the presence of three Aib-L-Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20-residue Peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane-perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage-gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence-specific 1H- and 13C-nmr assignments and conformational nmr parameters (3JNHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty-seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β-turns twisted into a continuous spiral from Asn2 to Leuol14 and forming a 26 A long helix-like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn2 side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φi = −90°, Ψi = −27°; Φi+1 = −98°, Ψi+1 = −17°; Φi+2 = −49°, Ψi+2 = −50°; Φi+3 = −78°, Ψi+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)n sequences, the (Xaa–Yaa–Aib–Pro)-β-bend ribbon spiral. It is stabilized by 4 1 intramolecular hydrogen bonds and differs from both the canonical 310-helix made of a succession of type III β-turns and from the β-bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999
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directed biosynthesis of Peptaibol antibiotics in two trichoderma strains ii structure elucidation
The Journal of Antibiotics, 1998Co-Authors: G Leclerc, Sylvie Rebuffat, Bernard BodoAbstract:Alpha-aminoisobutyric acid-directed biosynthesis in two Trichoderma strains has been shown to lead to the simplification of the natural Peptaibol microheterogeneous mixtures and to the production of new analogues. Hence, two new peptides originating from T. harzianum, trichorzin PA(U) 4 and harzianin PCU 4, were isolated by HPLC. Their sequences were determined by positive liquid secondary-ion mass spectrometry (LSI MS). Trichorzin PA(U) 4 and harzianin PC(U) 4 are 18- and 14-residue Peptaibols, respectively, both containing a high proportion of alpha-aminoisobutyric acid (Aib). LSI MS performed with lithium cationized peptides, allowed to assign the relative position of leucine/isoleucine isomeric residues, even without the use of tandem mass spectrometry.
Bernard Bodo - One of the best experts on this subject based on the ideXlab platform.
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Identification of Peptaibols from Trichoderma virens and cloning of a Peptaibol synthetase
The Journal of biological chemistry, 2002Co-Authors: Aric Wiest, Sylvie Rebuffat, Christophe Goulard, Bernard Bodo, Darlene Grzegorski, Daniel J. Ebbole, Charles M. KenerleyAbstract:The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high alpha-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a Peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virens was shown to produce a mixture of Peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all Peptaibol isoforms. Identification of the gene responsible for Peptaibol production will facilitate studies of the structure and function of Peptaibol antibiotics and their contribution to biocontrol activity.
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sequences and antimycoplasmic properties of longibrachins lgb ii and lgb iii two novel 20 residue Peptaibols from trichoderma longibrachiatum
Journal of Natural Products, 2001Co-Authors: G Leclerc, Christophe Goulard, Yann Prigent, Bernard Bodo, Henri Wroblewski, Sylvie RebuffatAbstract:Longibrachins are members of the class of natural Aib-containing peptides designated as Peptaibols. Six longibrachins, LGA I−IV and LGB II and III, were purified from a Trichoderma longibrachiatum strain by a procedure employing several chromatography steps including reversed-phase HPLC. The amino acid sequence determination was based on a combination of liquid secondary ion mass spectrometry (LSIMS) and two-dimensional 1H and 13C NMR spectroscopy. Longibrachins are 20-residue Peptaibols with a C-terminal phenylalaninol and either neutral (LGA; Gln18) or acidic (LGB; Glu18) character. Longibrachins LGB II and III have novel sequences. Both longibrachins LGA and LGB show significant bactericidal activity against mycoplasmas (Acholeplasma, Mycoplasma, and Spiroplasma), with minimal inhibitory concentrations in the range 1.56−12.5 μM (3−25 μg/mL), and also perturb the permeability of membrane bilayers. Longibrachin LGA IV is the most potent of the presently known 18−20-residue Peptaibols. The antimicrobial a...
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two unprecedented natural aib peptides with the xaa yaa aib pro motif and an unusual c terminus structures membrane modifying and antibacterial properties of pseudokonins kl iii and kl vi from the fungus trichoderma pseudokoningii
Journal of Peptide Science, 2000Co-Authors: Sylvie Rebuffat, Christophe Goulard, Sanae Hlimi, Bernard BodoAbstract:Pseudokonins KL III and KL VI are two natural ten-residue peptides, which both contain the (Xaa-Yaa-Aib-Pro) motif and exhibit an unusual C-terminus. They have been isolated from the fungus Trichoderma pseudokoningii by intensive reversed-phase HPLC, beside Peptaibols classically C-ended by a beta-amino alcohol. The amino acid sequences and the chemical structures of the C-ends have been determined by the combined use of positive ion LSI-MS and two-dimensional homo- and heteronuclear NMR, including COSY, TOCSY, ROESY, 13C heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC). Instead of one of the amino alcohols usually found as C-terminal residue in Peptaibols, pseudokonins KL III and KL VI are characterized by -Pro-NH2 and cyclo-(Aib-L-Proal) (Proal, prolinal), respectively. Such backbone modifications are described here for the first time for Peptaibol antibiotics. The unusual cyclo-(Aib-L-Proal) C-terminus is probably the result of an intramolecular cyclization of the two last Aib and Pro residues of a ten-amino acid precursor, via a Proal intermediate. A secondary structure stabilized by -C=O...H-N-hydrogen bonds of the 1<--4 type has been deduced for both peptides from ROESY data, 3JNHCalphaH couplings and amide proton temperature coefficient values. The (Xaa-Yaa-Aib-Pro) beta-bend ribbon spiral, which has been described for the first time in the case of a 14-residue Peptaibol containing three repetitive (Xaa-Yaa-Aib-Pro) motifs (Segalas G et al. Biopolymers 1999; 50: 71-85) appears to be maintained in the two shortened modified peptides. The beta-bend ribbon structure thus appears to be initiated by a single (Xaa-Yaa-Aib-Pro) motif and unaffected by the C-terminal modifications. However, the membrane and antibiotic properties of pseudokonins KL III and KL VI, point to the unfavourable effect of both shortening and cyclization of the peptide chain.
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characterization of a type of bend ribbon spiral generated by the repeating xaa yaa aib pro motif the solution structure of harzianin hc ix a 14 residue Peptaibol forming voltage dependent ion channels
Biopolymers, 1999Co-Authors: Isabelle Segalas, Bernard Bodo, Yann Prigent, Daniel Davoust, Sylvie RebuffatAbstract:The three-dimensional solution structure of harzianin HC IX, a Peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two-dimensional nmr spectroscopy combined with molecular modeling. This 14-residue peptide, Ac Aib1 Asn2 Leu3 Aib4 Pro5 Ala6 Ile7 Aib8 Pro9 Iva10 Leu11 Aib12 Pro13 Leuol14 (Aib, α-aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short-sequence Peptaibol class characterized by an acetylated N-terminus, a C-terminal amino alcohol, and the presence of three Aib-L-Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20-residue Peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane-perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage-gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence-specific 1H- and 13C-nmr assignments and conformational nmr parameters (3JNHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty-seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β-turns twisted into a continuous spiral from Asn2 to Leuol14 and forming a 26 A long helix-like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn2 side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φi = −90°, Ψi = −27°; Φi+1 = −98°, Ψi+1 = −17°; Φi+2 = −49°, Ψi+2 = −50°; Φi+3 = −78°, Ψi+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)n sequences, the (Xaa–Yaa–Aib–Pro)-β-bend ribbon spiral. It is stabilized by 4 1 intramolecular hydrogen bonds and differs from both the canonical 310-helix made of a succession of type III β-turns and from the β-bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999
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directed biosynthesis of Peptaibol antibiotics in two trichoderma strains ii structure elucidation
The Journal of Antibiotics, 1998Co-Authors: G Leclerc, Sylvie Rebuffat, Bernard BodoAbstract:Alpha-aminoisobutyric acid-directed biosynthesis in two Trichoderma strains has been shown to lead to the simplification of the natural Peptaibol microheterogeneous mixtures and to the production of new analogues. Hence, two new peptides originating from T. harzianum, trichorzin PA(U) 4 and harzianin PCU 4, were isolated by HPLC. Their sequences were determined by positive liquid secondary-ion mass spectrometry (LSI MS). Trichorzin PA(U) 4 and harzianin PC(U) 4 are 18- and 14-residue Peptaibols, respectively, both containing a high proportion of alpha-aminoisobutyric acid (Aib). LSI MS performed with lithium cationized peptides, allowed to assign the relative position of leucine/isoleucine isomeric residues, even without the use of tandem mass spectrometry.
Yuzhong Zhang - One of the best experts on this subject based on the ideXlab platform.
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the putative methyltransferase tllae1 is involved in the regulation of Peptaibols production in the biocontrol fungus trichoderma longibrachiatum smf2
Frontiers in Microbiology, 2020Co-Authors: Jinchao Shi, Xiulan Chen, Yuzhong Zhang, Weiling Shi, Yanrong Zhou, Xia Zhang, Weixin Zhang, Xiao-yan SongAbstract:The biocontrol fungus Trichoderma longibrachiatum SMF2 secretes a large quantity of Peptaibols that have been shown to have a range of biological activities and therefore great application values. However, the mechanism of the regulatory expression of Peptaibols is still unclear. The putative methyltransferase LaeA/LAE1 is a global regulator involved in the biosynthesis of some secondary metabolites in filamentous fungi. In this study, we demonstrated that the ortholog of LaeA/LAE1 in the biocontrol fungus T. longibrachiatum SMF2, TlLAE1, plays an important role in the regulation of Peptaibols production. Deletion of Tllae1 resulted in a slight negative impact on mycelial growth, and a significant defect in conidial production. Deletion of Tllae1 also compromised the production of Peptaibols to a large degree. Further analyses indicated that this defect occurred at the transcriptional level of the two synthetases-encoding genes, tlx1 and tlx2, which are responsible for Peptaibols production. By contrast, constitutive expression of Tllae1 in T. longibrachiatum SMF2 led to 2-fold increased Peptaibols production, suggesting that this is a strategy to improve Peptaibols production in Trichoderma fungi. These results demonstrate the important role of LAE1 in the regulation of Peptaibols production in T. longibrachiatum SMF2.
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trichokonins from trichoderma pseudokoningii smf2 induce resistance against gram negative pectobacterium carotovorum subsp carotovorum in chinese cabbage
Fems Microbiology Letters, 2014Co-Authors: Xiulan Chen, Yuzhong Zhang, Lei-lei Chen, Haiyun Li, Xiusheng Zhang, Zhiting Gong, Xiao-yan SongAbstract:: Peptaibols, mainly produced by Trichoderma, play a pivotal role in controlling plant disease caused by fungi, virus, and Gram-positive bacteria. In the current study, we evaluated the control effect of Trichokonins, antimicrobial Peptaibols from Trichoderma pseudokoningii SMF2, on soft rot disease of Chinese cabbage caused by a Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum and analyzed the mechanism involved. Trichokonins treatment (0.3 mg L(-1) ) enhanced the resistance of Chinese cabbage against Pcc infection. However, Trichokonins could hardly inhibit the growth of Pcc in vitro, even at high concentration (500 mg L(-1) ). Therefore, the direct effect of Trichokonins on Pcc may not the main reason why Trichokonins could control soft rot of Chinese cabbage. Trichokonin treatment led to an obvious increase in the production of reactive oxygen species hydrogen peroxide and superoxide radical, a significant enhance of the activities of pathogenesis-related enzymes catalase, polyphenoloxidase and peroxidase, and upregulation of the expression of salicylic acid - responsive pathogenesis-related protein gene acidic PR-1a in Chinese cabbage. These results indicate that Trichokonins induce resistance in Chinese cabbage against Pcc infection through the activation of salicylic acid signaling pathway, which imply the potential of Trichoderma and Peptaibols in controlling plant disease caused by Gram-negative bacteria.
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antimicrobial Peptaibols induce defense responses and systemic resistance in tobacco against tobacco mosaic virus
Fems Microbiology Letters, 2010Co-Authors: Yan Luo, Xiulan Chen, Xiao-yan Song, Lei-lei Chen, Dandan Zhang, Xiaowei Dong, Peibao Zhao, Xingjun Wang, Mei Shi, Yuzhong ZhangAbstract:Trichoderma spp. are well-known biocontrol agents because of their antimicrobial activity against bacterial and fungal phytopathogens. However, the biochemical mechanism of their antiviral activity remains largely unknown. In this study, we found that Trichokonins, antimicrobial Peptaibols isolated from Trichoderma pseudokoningii SMF2, could induce defense responses and systemic resistance in tobacco (Nicotiana tabacum var. Samsun NN) against tobacco mosaic virus (TMV) infection. Local Trichokonin (100 nM) treatment led to 54% lesion inhibition, 57% reduction in average lesion diameter and 30% reduction in average lesion area in systemic tissue of tobacco compared with control, indicating that Trichokonins induced resistance in tobacco against TMV infection. Trichokonin treatment increased the production of reactive oxygen species and phenolic compounds in tobacco. Additionally, application of Trichokonins significantly increased activities of pathogenesis-related enzymes PAL and POD, and upregulated the expression of several plant defense genes. These results suggested that multiple defense pathways in tobacco were involved in Trichokonin-mediated TMV resistance. We report on the antivirus mechanism of Peptaibols, which sheds light on the potential of Peptaibols in plant viral disease control.
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solid state fermentation for trichokonins production from trichoderma koningii smf2 and preparative purification of trichokonin vi by a simple protocol
Journal of Biotechnology, 2007Co-Authors: Xiao-yan Song, Xiulan Chen, Yuzhong ZhangAbstract:Trichokonins are Peptaibols produced by Trichoderma koningii SMF2. The main isoforms are Trichokonin VI, Trichokonin VII and Trichokonin VIII. The solid-state fermentation (SSF) was applied for the production of Trichokonin VI. The fermentation factors, which included inoculum size, incubation temperature, initial moisture content and initial pH, were investigated and optimized by response surface methodology. The maximum Trichokonin VI production (4.07 mg/g dry substrate) was achieved by employing inoculum size of 18%, incubation temperature at 24.3 °C, initial moisture content of 77.5% and initial pH at 5.0. Furthermore, gel filtration and preparative HPLC were used for separation of Trichokonin VI from a crude extract of the T. koningii SMF2 culture. With this preparative purification protocol under optimized fermentation conditions, 146.20 mg Trichokonin VI was obtained from 1 kg solid cultures. It has been shown that the obtained Trichokonin VI is more than 95% in purity. This is the first report on optimization of Peptaibols production in SSF with high content. An efficient method for the preparative purification of Trichokonin VI is also proposed.
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isolation purification and identification of three Peptaibols from trichoderma koningii with antibiotic activity against ralstonia solancearum
Journal of Zhejiang University Agriculture and Life Sciences, 2004Co-Authors: Qingtao Shen, Xiulan Chen, Yuzhong ZhangAbstract:The use of microorganisms for biological purposes has become an effective alternative to control plant pathogens. Trichoderma koningii Smf2 was chosen from eight Trichoderma strains for its thermostatic metabolites with antibiotic activity against Ralstonia solancearum Smith. Exclusion chromatography (LH20) was used twice to partially purify targeted metabolites combined with biological test. LC/ESI-MS, a powerful tool for rapid identification and sequence determination peptides, identified these metabolites as three Peptaibols named Trichokonin VI, VII and VIII, and their sequences were confirmed with NMR.
Charles M. Kenerley - One of the best experts on this subject based on the ideXlab platform.
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Two classes of new Peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens.
The Journal of biological chemistry, 2010Co-Authors: Prasun K. Mukherjee, Aric Wiest, Nicolas Ruiz, Andrew Keightley, Maria E. Moran-diez, Kevin Mccluskey, Yves François Pouchus, Charles M. KenerleyAbstract:Peptaibols are a group of small peptides having a high α-aminoisobutyric acid (Aib) content and produced by filamentous fungi, especially by the members of the genus Trichoderma (anamorph Hypocrea). These antibiotics are economically important for their anti-microbial and anti-cancer properties as well as ability to induce systemic resistance in plants against microbial invasion. In this study we present sequences of two classes (11-residue and 14-residue) of Peptaibols produced by the biocontrol fungus Trichoderma virens. Of the 35 11-residue Peptaibols sequenced, 18 are hitherto not described, and all the 53 14-residue sequences described by us here are new. We have also identified a Peptaibol synthetase (non-ribosomal peptide synthetase, NRPS) with 14 complete modules in the genome of this fungus and disruption of this single gene (designated as tex2) resulted in the loss of both the classes of Peptaibols. We, thus present here an unprecedented case where a single NRPS encodes for two classes of Peptaibols. The new Peptaibols identified here could have applications as therapeutic agents for the management of human and plant health.
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Identification of Peptaibols from Trichoderma virens and cloning of a Peptaibol synthetase
The Journal of biological chemistry, 2002Co-Authors: Aric Wiest, Sylvie Rebuffat, Christophe Goulard, Bernard Bodo, Darlene Grzegorski, Daniel J. Ebbole, Charles M. KenerleyAbstract:The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high alpha-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a Peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virens was shown to produce a mixture of Peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all Peptaibol isoforms. Identification of the gene responsible for Peptaibol production will facilitate studies of the structure and function of Peptaibol antibiotics and their contribution to biocontrol activity.
Christophe Goulard - One of the best experts on this subject based on the ideXlab platform.
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Identification of Peptaibols from Trichoderma virens and cloning of a Peptaibol synthetase
The Journal of biological chemistry, 2002Co-Authors: Aric Wiest, Sylvie Rebuffat, Christophe Goulard, Bernard Bodo, Darlene Grzegorski, Daniel J. Ebbole, Charles M. KenerleyAbstract:The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high alpha-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a Peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virens was shown to produce a mixture of Peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all Peptaibol isoforms. Identification of the gene responsible for Peptaibol production will facilitate studies of the structure and function of Peptaibol antibiotics and their contribution to biocontrol activity.
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sequences and antimycoplasmic properties of longibrachins lgb ii and lgb iii two novel 20 residue Peptaibols from trichoderma longibrachiatum
Journal of Natural Products, 2001Co-Authors: G Leclerc, Christophe Goulard, Yann Prigent, Bernard Bodo, Henri Wroblewski, Sylvie RebuffatAbstract:Longibrachins are members of the class of natural Aib-containing peptides designated as Peptaibols. Six longibrachins, LGA I−IV and LGB II and III, were purified from a Trichoderma longibrachiatum strain by a procedure employing several chromatography steps including reversed-phase HPLC. The amino acid sequence determination was based on a combination of liquid secondary ion mass spectrometry (LSIMS) and two-dimensional 1H and 13C NMR spectroscopy. Longibrachins are 20-residue Peptaibols with a C-terminal phenylalaninol and either neutral (LGA; Gln18) or acidic (LGB; Glu18) character. Longibrachins LGB II and III have novel sequences. Both longibrachins LGA and LGB show significant bactericidal activity against mycoplasmas (Acholeplasma, Mycoplasma, and Spiroplasma), with minimal inhibitory concentrations in the range 1.56−12.5 μM (3−25 μg/mL), and also perturb the permeability of membrane bilayers. Longibrachin LGA IV is the most potent of the presently known 18−20-residue Peptaibols. The antimicrobial a...
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two unprecedented natural aib peptides with the xaa yaa aib pro motif and an unusual c terminus structures membrane modifying and antibacterial properties of pseudokonins kl iii and kl vi from the fungus trichoderma pseudokoningii
Journal of Peptide Science, 2000Co-Authors: Sylvie Rebuffat, Christophe Goulard, Sanae Hlimi, Bernard BodoAbstract:Pseudokonins KL III and KL VI are two natural ten-residue peptides, which both contain the (Xaa-Yaa-Aib-Pro) motif and exhibit an unusual C-terminus. They have been isolated from the fungus Trichoderma pseudokoningii by intensive reversed-phase HPLC, beside Peptaibols classically C-ended by a beta-amino alcohol. The amino acid sequences and the chemical structures of the C-ends have been determined by the combined use of positive ion LSI-MS and two-dimensional homo- and heteronuclear NMR, including COSY, TOCSY, ROESY, 13C heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC). Instead of one of the amino alcohols usually found as C-terminal residue in Peptaibols, pseudokonins KL III and KL VI are characterized by -Pro-NH2 and cyclo-(Aib-L-Proal) (Proal, prolinal), respectively. Such backbone modifications are described here for the first time for Peptaibol antibiotics. The unusual cyclo-(Aib-L-Proal) C-terminus is probably the result of an intramolecular cyclization of the two last Aib and Pro residues of a ten-amino acid precursor, via a Proal intermediate. A secondary structure stabilized by -C=O...H-N-hydrogen bonds of the 1<--4 type has been deduced for both peptides from ROESY data, 3JNHCalphaH couplings and amide proton temperature coefficient values. The (Xaa-Yaa-Aib-Pro) beta-bend ribbon spiral, which has been described for the first time in the case of a 14-residue Peptaibol containing three repetitive (Xaa-Yaa-Aib-Pro) motifs (Segalas G et al. Biopolymers 1999; 50: 71-85) appears to be maintained in the two shortened modified peptides. The beta-bend ribbon structure thus appears to be initiated by a single (Xaa-Yaa-Aib-Pro) motif and unaffected by the C-terminal modifications. However, the membrane and antibiotic properties of pseudokonins KL III and KL VI, point to the unfavourable effect of both shortening and cyclization of the peptide chain.
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the Peptaibol antibiotics from trichoderma soil fungi structural diversity and membrane properties
1999Co-Authors: S Rebuffat, Christophe Goulard, B. Bodo, M F RequebertAbstract:Peptide antibiotics of the Peptaibol class are 7- to 20-residue linear peptides produced by Trichoderma soil fungi. They have blocked N-and C-termini and contain a high proportion of α,α-dialkylated amino acids, such as α-aminoisobutyric acid (Aib). They exhibit remarkable membrane-perturbing properties. The present review focuses on our recent results on Peptaibols in the context of the studies developed overworld. The three main sub-classes of Peptaibols, i.e. long-sequence, short-sequence and lipoPeptaibols exhibit special structural characteristics that are presented. We describe the three-dimensional structures established for the different sub-classes, either in methanol solution or in the presence of micelles. We point particularly to a new type of helix that we recently described and that occurs with the repeating (Xaa Yaa Aib Pro) amino acid motif encountered in short-sequence Peptaibols. To stress the three-dimensional structure / membrane properties relationship for the three Peptaibol sub-classes, the liposome permeabilization properties and voltage-gated channel formation ability are described in relation with the antimicrobial activity. The perspectives of research in this field are discussed.
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directed biosynthesis of Peptaibol antibiotics in two trichoderma strains i fermentation and isolation
The Journal of Antibiotics, 1998Co-Authors: G Leclerc, Sylvie Rebuffat, Christophe Goulard, Bernard BodoAbstract:Peptaibols are linear α-aminoisobutyric acid-containing peptide antibiotics originating from soil fungi mainly of the genus Trichoderma and biosynthesized in complex mixtures of closely related analogues by a polyenzymatic pathway. Addition of amino acids such as α-aminoisobutyric acid (Aib), glutamic acid or arginine, to the fermentation medium of two Trichoderma strains, T. harzianum and T. longibrachiatum, has been shown to result in the simplification of the natural Peptaibol mixtures, leading in each case to the almost exclusive biosynthesis of a single peptide. Surprisingly, the obtained peptides are Aib-enriched, whether the added amino acid is Aib, Glu or Arg. By adding Aib to the fermentation medium of T. harzianum, two new Aib-rich Peptaibols were isolated. Moreover, adding glutamic acid to the culture medium of T. longibrachiatum, which produces both neutral and acidic 20-residue Peptaibols with either glutamine or glutamic acid at position 18, increases the production of the acidic peptides. However, arginine which is a positively charged amino acid generally absent from Peptaibol sequences, is not incorporated in trichorzins when added to the fermentation medium of T. harzianum.