Detection of putative peptide synthetase genes in Trichoderma species: application of this method to the cloning of a gene from T. harzianum CECT 2413

Some of the secondary metabolites produced by Trichoderma, such as the peptaibols and other antibiotics, have a peptide structure and in their biosynthesis are involved proteins belonging to the Non-Ribosomal Peptide Synthetase family. In the present work, a PCR-mediated strategy was used to clone a region corresponding to an adenylation domain of a peptide synthetase (PS) gene from 10 different strains of Trichoderma. In addition, and using the fragment isolated by PCR from T. harzianum CECT 2413 as a probe, a fragment of 19.0 kb corresponding to a PS-encoding gene named salps1, including a 1.5 kb fragment of the promoter, was cloned and sequenced. The cloned region of salps1 contains four complete, and a fifth incomplete, modules, in which are found the adenylation, thiolation and condensation domains, but also an additional epimerization domain at the C-terminal end of the first module. The analysis of the Salps1 protein sequence, taking into consideration published data, suggests that it is neither a peptaibol synthetase nor a protein involved in siderophore biosynthesis. The presence of two breaks in the open reading frame and the expression of this gene under nitrogen starvation conditions suggest that salps1 could be a pseudogene.     

Identification and cloning of peptide synthetase genes of thermostable bacilli using the polymerase chain reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genes dhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Identification of peptaibols from Trichoderma virens and cloning of a peptaibol synthetase

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.     

Isolation and characterisation of a partial peptide synthetase gene from Trichoderma asperellum

Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a lambda FIXII genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1.     

Identification and Cloning of Peptide Synthetase Genes of Thermostable Bacilli Using the Polymerase Chain Reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genesdhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases

Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes. Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity). The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid. We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation. We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences. Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained.     

Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus

Little is known about the process whereby the emetic toxin (or cereulide) of Bacillus cereus is produced. Two cereulide-producing strains of B. cereus were cloned and sequenced following polymerase chain reaction (PCR) amplification with primers that were specific for conserved regions of non-ribosomal peptide synthetase (NRPS) genes. The cloned regions of the B. cereus strains were highly homologous to conserved regions of other peptide synthetase nucleotide sequences. Primers were designed for two variable regions of the NRPS gene sequence to ensure specificity for the emetic strains. A total of 86 B. cereus strains of known emetic or non-emetic activity were screened using these primers. All of the emetic strains (n=30) displayed a 188 bp band following amplification and gel electrophoresis. We have developed an improved method of identifying emetic strains of B. cereus and provided evidence that cereulide is produced by peptide synthetases.     

Cloning and sequence analysis of an intron-containing domain from a peptide synthetase-encoding gene of the entomopathogenic fungus Metarhizium anisopliae

A gene encoding a putative peptide synthetase has been cloned and partially sequenced from the filamentous fungus, Metarhizium anisopliae. The deduced amino acid sequence of one entire domain and the following spacer is typical of fungal peptide synthetases, showing good conservation of the six expected core sequences. There are two introns within this region, the first interrupting core 5 (RLDLTDIE) of the domain and the second in a conserved area of the spacer region.     

Sequence analysis and modular organization of threonyl-tRNA synthetase from Thermus thermophilus and its interrelation with threonyl-tRNA synthetases of other origins.

The gene encoding threonyl-tRNA synthetase (Thr-tRNA synthetase) from the extreme thermophilic eubacterium Thermus thermophilus HB8 has been cloned and sequenced. The ORF encodes a polypeptide chain of 659 amino acids (Mr 75 550) that shares strong similarities with other Thr-tRNA synthetases. Comparative analysis with the three-dimensional structure of other subclass IIa synthetases shows it to be organized into four structural modules: two N-terminal modules specific to Thr-tRNA synthetases, a catalytic core and a C-terminal anticodon-binding module. Comparison with the three-dimensional structure of Escherichia coli Thr-tRNA synthetase in complex with tRNAThr enabled identification of the residues involved in substrate binding and catalytic activity. Analysis by atomic absorption spectrometry of the enzyme overexpressed in E. coli revealed the presence in each monomer of one tightly bound zinc atom, which is essential for activity. Despite strong similarites in modular organization, Thr-tRNA synthetases diverge from other subclass IIa synthetases on the basis of their N-terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, that are not significantly similar to the shorter extension of the archaebacterial enzymes. Investigation of a truncated Thr-tRNA synthetase demonstrated that domain N1 is not essential for tRNA charging. Thr-tRNA synthetase from T. thermophilus is of the eubacterial type, in contrast to other synthetases from this organism, which exhibit archaebacterial characteristics. Alignments show conservation of part of domain N2 in the C-terminal moiety of Ala-tRNA synthetases. Analysis of the nucleotide sequence upstream from the ORF showed the absence of both any anticodon-like stem-loop structure and a loop containing sequences complementary to the anticodon and the CCA end of tRNAThr. This means that the expression of Thr-tRNA synthetase in T. thermophilus is not regulated by the translational and trancriptional mechanisms described for E. coli thrS and Bacillus subtilis thrS and thrZ. Here we discuss our results in the context of evolution of the threonylation systems and of the position of T. thermophilus in the phylogenic tree.     

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains.

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.     

Multiple non-ribosomal peptide synthetase genes determine peptaibol synthesis in Trichoderma virens

Trichoderma virens, an imperfect fungus, is used as a biocontrol agent to suppress plant disease caused by soilborne fungal pathogens. Antimicrobial peptides it produces include peptaibols of 11, 14, and 18 amino acids in length. These peptaibols were previously reported to be synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by the Tex1 gene in strain Tv29-8. The present study examined the Tex1 homolog in a commercially relevant T. virens strain, G20. Although the gene in G20 was 99% identical in DNA sequence to Tex1 in the 15.8 kb compared, gene disruption results indicate that it is only responsible for the production of an 18-mer peptaibol, and not 11-mer and 14-mer peptaibols. Additional NRPS adenylate domains were identified in T. virens and one was found to be part of a 5-module NRPS gene. Although the multimodule gene is not needed for peptaibol synthesis, sequence comparisons suggest that two of the individual adenylate domain clones might be part of a separate peptaibol synthesis NRPS gene. The results indicate a significant diversity of NRPS genes in T. virens that is unexpected from the literature.     

The production of multiple small peptaibol families by single 14-module Peptide synthetases in Trichoderma/Hypocrea

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

A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).     

Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90

Anabaena strain 90 produces three hepatotoxic heptapeptides (microcystins), two seven-residue depsipeptides called anabaenopeptilide 90A and 90B, and three six-residue peptides called anabaenopeptins. The anabaenopeptilides belong to a group of cyanobacterial depsipeptides that share the structure of a six-amino-acid ring with a side-chain. Despite their similarity to known cyclic peptide toxins, no function has been assigned to the anabaenopeptilides. Degenerate oligonucleotide primers based on the conserved amino acid sequences of other peptide synthetases were used to amplify DNA from Anabaena 90, and the resulting polymerase chain reaction (PCR) products were used to identify a peptide synthetase gene cluster. Four genes encoding putative anabaenopeptilide synthetase domains were characterized. Three genes, apdA, apdB and apdD, contain two, four and one module, respectively, encoding a total of seven modules for activation and peptide bond formation of seven L-amino acids. Modules five and six also carry methyltransferase-like domains. Before the first module, there is a region similar in amino acid sequence to formyltransferases. A fourth gene (apdC), between modules six and seven, is similar in sequence to halogenase genes. Thus, the order of domains is co-linear with the positions of amino acid residues in the finished peptide. A mutant of Anabaena 90 was made by inserting a chloramphenicol resistance gene into the apdA gene. DNA amplification by PCR confirmed the insertion. Mass spectrometry analysis showed that anabaenopeptilides are not made in the mutant strain, but other peptides, such as microcystins and anabaenopeptins, are still produced by the mutant.     

Cloning and sequencing of the gltX gene, encoding the glutamyl-tRNA synthetase of Rhizobium meliloti A2.

The gltX gene, coding for the glutamyl-tRNA synthetase of Rhizobium meliloti A2, was cloned by using as probe a synthetic oligonucleotide corresponding to the amino acid sequence of a segment of the glutamyl-tRNA synthetase. The codons chosen for this 42-mer were those most frequently used in a set of R. meliloti genes. DNA sequence analysis revealed an open reading frame of 484 codons, encoding a polypeptide of Mr 54,166 containing the amino acid sequences of an NH2-terminal and various internal fragments of the enzyme. Compared with the amino acid sequence of the glutamyl-tRNA synthetase of Escherichia coli, the N-terminal third of the R. meliloti enzyme was strongly conserved (52% identity); the second third was moderately conserved (38% identity) and included a few highly conserved segments, whereas no significant similarity was found in the C-terminal third. These results suggest that the C-terminal part of the protein is probably not involved in the recognition of substrates, a feature shared with other aminoacyl-tRNA synthetases.     

Two classes of new peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens

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.     

Cloning, sequencing, and characterization of the iturin A operon

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.     

A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding

Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20-70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNA(Asp), we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identifies a lysine-rich 11 residue sequence ((29)LSKKALKKLQK(39) in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide.