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Moulds and yeasts are frequently referred as microbial contaminants of feed meals used in intensive animal production. Most of the sanitary risks that are present in milk, eggs and meat are related with the safety of animal feeds. In this study, 75 samples of swine feed, being 10 feed meals and 65 granulated, were tested for mycological characterisation, using conventional methods (NP-3277-2; 2002). Only two granulated feed were negative (2.7%). Out of 75 samples, 73 (97.3%) were positive. Mean count of fungi has been 6.6 × 10² cfu/g ranging from 2.7 × 10¹ to 2.7 × 10³ cfu/g; yeasts were present in 69.9% of the positive samples. Potential toxigenic moulds (Fusarium spp., Aspergillus flavus and Penicillium spp.) were present in all the positive samples with mean levels of 3.2 log₁₀ cfu/g, 2.8 log₁₀ cfu/g and 3.0 log₁₀ cfu/g, respectively. Other genera found were Phoma, Rhizopus and Paecillomyces, with low levels of contamination (32.9%, 35.6% and 47.9%, respectively). It was concluded that the levels and frequency of mycobiota contamination are decreasing judging the results obtained in the last ten years, in Portugal.

An important aspect to consider in the modulation of gene expression with biotechnological purposes is mRNA stability. The KlCYC1 gene has a long (1.2 kb) 3′-UTR region that can be used to modulate gene expression in yeast by the alternative use of its proximal or distal 3′-Untranslated Region [1, 2]. The stability of the two KlCYC1 transcripts was analysed in Saccharomyces cerevisiae puf3 and rpb1-1 mutants. When the puf3 mutant and the deletion of the UGUR element at positions (131-135) were combined, there was a two-fold increase in total KlCYC1 levels mainly due to the increase in the long transcript signal. After a cease of transcription (rpb1-1 mutant), the long transcript was stable for more than two hours while the short one for less than one. When the gene was expressed in the yeast Kluyveromyces lactis under hypoxic conditions, both transcripts were degraded faster than in the rpb1-1 mutant. These findings suggest the presence of different mRNA turnover mechanisms able to operate on KlCYC1 transcripts under different physiological conditions.

The gene HIS4 from Kluyveromyces lactis is transcriptionally activated in complete synthetic respect to rich media and in an independent mechanism related to carbon source. This regulation was not previously described for Saccharomyces cerevisiae HIS4. The EMSA assay carried out with F7 showed a specific band, Fc1, in YPG, and two bands, Fc2 and Fc3, in complete medium. The Fc2 and Fc3 bands were dependent on the carbon source present in the medium, since their intensities were higher in glycerol than in glucose. The protein or proteins causing the Fc1 band seem to be involved in the different regulation mechanisms between rich and synthetic complete media because the Fc1 band was detected in cells grown in synthetic medium. Therefore, the promoter region (-200 to -173) is responsible for two independent regulatory mechanisms.

The population dynamics of yeasts in oleic ecosystems in the Castilla la Mancha region (Spain) was analysed for two consecutive years (2007 to 2008) in two different varieties (Cornicabra and Arbequina respectively). Yeasts were isolated from of fresh olives (Olea europaea L.) fruits, olive pomace (solid waste) and olive paste (crush olives). All yeast species were identified by RFLPs of their rDNA but in some species sequence analysis of the 5.8S rDNA gene was necessary. This study allowed to identify 108 yeast isolated which belonging to seven different genera (Zygosaccharomyces, Pichia, Lachancea, Kluyveromyces, Saccharomyces, Candida, Torulaspora) and fourteen species. The most representative species were Pichia caribbica, Zygosaccharomyces fermentati and Pichia holstii. The yeast characterization was studied by means of several enzymes how β-glucosidase, β-glucanase, carboxymethylcellulase, polygalacturonase, peroxydase and lipase and was observed that the mayority of them presented β-glucanase, β-glucosidase and peroxidase activities, a few had cellulase and polygalacturonase activities and none of species showed lipase activity.

We have developed a 2-day lab-practice to present microbiology and biotechnology to science secondary school students. This initiative seeks to familiarize students with microbial physiology and fermentation technology and to stimulate debate between students and researchers. This project has strengthened the relationship between secondary school science (students and teachers) and university teachers and researchers.

The YARE (Yeast AP1-Recognition Element) is the DNA binding site for YAp1 and YAp2 transcriptional activators in the presence of cadmium. The roles of three sequences matching the YARE consensus on the KlHIS4 promoter are analysed to determine their implication in cadmium transcriptional regulation. A specific band, Cd2, is characterised by EMSA in the region encompassing positions -283 to -276, which disappears in the presence of cadmium or when the YARE consensus is mutated. The factor causing Cd2 is named CSP1 (Cadmium Sensitive Protein 1). The binding of a second factor (P3) producing the band Cd3, also dependent on the YARE consensus, but insensitive to cadmium, is also characterised in the same region. The gel-shift pattern of region -384 to -323 with no YARE consensus is also altered by cadmium, thus, the cadmium transcriptional response of KlHIS4 appears to be modulated by multiple promoter elements. The presence of YARE regulatory elements is not a guarantee of a specific cadmium transcriptional regulation in K. lactis.

Two genomic fragments containing the KlADE2 and the KlPUT2 genes were isolated from Kluyveromyces lactis genomic library by functional complementation of the corresponding ade2 and put2 mutations in Saccharomyces cerevisiae. The deduced KlAde2p and KlPut2p amino acid sequences displayed strong similarities to their counterparts in Saccharomyces cerevisiae (75% and 72% identity respectively) with highly conserved domains. Thus, we report the isolation and characterization of ADE2 and PUT2 genes from Kluyveromyces lactis, including their protein structure, flanking sequence regions, and transcriptional gene regulation by different nutrients in the medium.

Cell membranes have the ability to bend and curve, thus providing clathrine-coated pits and plasmalemma caveolae, and facilitating many cell functions such as receptor-mediated endocytosis. On the other hand all intracellular membranes are highly deformable, producing cargo vesicles destined to organelles and plasma membrane. Generation of membrane curvature is currently believed to involve the penetration of amphipathic helix into the cytosolic face of the membrane bilayer, producing an asymmetry between the two membrane leaflets and generating bending and curvature towards the cytosol. Here we show, using thin section and freeze-fracture electron microscopy, that ethidium bromide is able to produce negative curvature toward the cytosol in Candida utilis yeast cells. The curvatures were produced in grooves area, resulting in cup-shaped structures with centrally located groove or in polymorph structures with laterally located grooves; these structures were termed "nanocups". Apparently the curvatures were not able to generate vesicles and tubules, suggesting that they were not involved in intracellular trafficking. Thus besides mechanically- or biologically-produced curvature, we can add chemically-produced curvature the function of which remains to be elucidated.