Detection of peptaibols and partial cloning of a putative peptaibol synthetase gene from T. harzianum CECT 2413.

The characterization of 11- and 18-residue peptaibols (peptides synthesized by peptide synthetases) at Trichoderma harzianum CECT 2413 (a filamentous fungus) was performed. Using a heterologous probe from tex1, the only peptaibol synthetase cloned and characterized so far in Trichoderma species, was cloned; a region that comprised 11676 bp of a second peptide synthetase gene detected in these strain (called salps2) and sequenced. The deduced sequence of Salps2 (3891 amino acids) contained three complete and a fourth incomplete module of a peptide synthetase, in which the typical adenylation, thiolation and condensation domains were found, but also an additional dehydrogenase/reductase domain in the C-terminus of the last module. Based on sequence similarity and analysis of its modular structure, it is proposed that Salps2 is a peptaibol synthetase. Additionally, analysis of =4.4-kb sequence downstream of salps2 was done and the signature sequences of Salps2 were identified and compared with those of available sequences of the other Trichoderma peptaibol synthetases.

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.

Colour pigments of Trichoderma harzianum. Preliminary investigations with thin-layer chromatography-Fourier transform infrared spectroscopy and high-performance liquid chromatography with diode array and mass spectrometric detection.

The colour pigments of Trichoderma harzianum fermentation broth were separated and the main fractions were tentatively identified by reversed-phase thin-layer chromatography-Fourier transform infrared spectroscopy (RP-TLC-FT-IR), RP-HPLC-diode array detection and RP-HPLC-MS. It was established that the multistep gradient elution developed for RP-TLC separation of pigments can be successfully used as a pilot method for the rational design of gradient elution in RP-HPLC for the separation of the same pigments. FT-IR and MS measurements were unable to identify the exact chemical structures of the main pigment fractions, the presence of OH, =CH and C=O (RP-TLC-FT-IR) and OH and NH, substructures (RP-HPLC-MS) was confirmed. It was assumed that the main pigment fractions are oxidation polymers originating from monomer molecules containing polar substructures and double bonds in the alkyl chain which are liable for oxidation during the aerobic fermentation process.

Microbial contamination of carcasses and equipment from an Iberian pig slaughterhouse.

The microbial contamination of carcasses and equipment has been studied in an industrial slaughterhouse of Iberian pigs. Samples of the surface of carcasses were taken at different stages of the process and aerobic plate count at 37 degrees C (APC), Enterobacteriaceae-count (E-count) and Escherichia coli-count (EC-count) were determined. It was demonstrated that in scalding and singeing the APC decreased (P < 0.01), while in the dehairing it increased (P < 0.01). The E-count and EC-count decreased in the scalding but increased in the evisceration (P < 0.001). The implementation of good manufacturing practices (GMP) in the stages of closure of the anus and evisceration significantly decreased the EC-count. It changed from 61.1% in carcasses without GMP that had counts higher than 1 log CFU/cm2 to only 7.4% in GMP carcasses. A final wash of the carcasses with potable water at high pressure (the only decontaminating treatment permitted in the European Union) was tested and failed to decrease the counts. It was also demonstrated that cleaning and disinfection of the dehairing and scraping machines is not effective.

Separation and identification of volatile components in the fermentation broth of Trichoderma atroviride by solid-phase extraction and gas chromatography-mass spectrometry.

A preseparated fermentation broth of Trichoderma atroviride strain 11 is analyzed by gas chromatography followed by mass-spectral detection using a Finnigan MAT GCQ apparatus. After preseparation in a C18 and a silica gel column, nineteen pyrone and dioxolane derivatives and two aliphatic esters are obtained, respectively. Among these, the four dioxolane derivatives have not been identified previously. The main component is found to be 5,5'-dimethyl-2H-pyran-2-on. The relative standard deviation for the determination of the retention time and the peak area (measured in ion counts) is 0.1% and 4.5%, respectively.

Cloning and characterization of the erg1 gene of Trichoderma harzianum: effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine.

Trichoderma species are commonly used as biocontrol agents of different plant-pathogenic fungi. Terpene compounds are involved in the biocontrol process due to their antifungal properties (e.g., ergokonins and viridins) but additionally their structural function in the cell membranes (ergosterol) is essential. We report here the characterization of the T. harzianum erg1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives such as ergosterol. In T. harzianum the partial silencing of the erg1 gene gave rise to transformants with a higher level of sensitivity to terbinafine, an antifungal compound that acts specifically over the squalene epoxidase activity. In addition, these silenced transformants produced lower levels of ergosterol than the wild type strain. Finally, the silencing of the erg1 gene resulted in an increase in the expression level of the erg7 gene that encodes the oxidosqualene lanosterol-cyclase, another enzyme of the terpene biosynthesis pathway.

ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum.

The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu-Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.

Riesgo de alcoholismo y accidentalidad laboral en trabajadores del sector de la construcción en Santafé de Bogotá D.C., 1996 / Alcoholism and labor accident risk in construction workers in Bogotá, 1996

LAS HIPÓTESIS PLANTEADAS FUERON: Existe una mayor frecuencia de accidentes de trabajo en los trabajadores con alto grado de alcoholismo, comparado contra trabajadores con bajo riesgo de alcoholismo. Existe una variedad más alta en las consecuencias negativas de los accidentes de trabajo en los trabajadores con alto riesgo de alcoholismo en relación. De acuerdo a los resultados del estudio se recomienda a las empresas de la construcción implementar una política clara sobre el consumo de bebidas alcohólicas en el ámbito laboral y diseñar y ejecutar un programa de prevención del alcoholismo.