When treated with a hyperosmotic stimulus, Kluyveromyces lactis cells respond by activating the mitogen-activated protein kinase (MAPK) K. lactis Hog1 (KlHog1) protein via two conserved branches, SLN1 and SHO1. Mutants affected in only one branch can cope with external hyperosmolarity by activating KlHog1p by phosphorylation, except for single ⌬Klste11 and ⌬Klste50 mutants, which showed high sensitivity to osmotic stress, even though the other branch (SLN1) was intact. Inactivation of both branches by deletion of KlSHO1 and KlSSK2 also produced sensitivity to high salt. Interestingly, we have observed that in ⌬Klste11 and ⌬Klsho1 ⌬Klssk2 mutants, which exhibit sensitivity to hyperosmotic stress, and contrary to what would be expected, KlHog1p becomes phosphorylated. Additionally, in mutants lacking both MAPK kinase kinases (MAPKKKs) present in K. lactis (KlSte11p and KlSsk2p), the hyperosmotic stress induced the phosphorylation and nuclear internalization of KlHog1p, but it failed to induce the transcriptional expression of KlSTL1 and the cell was unable to grow in high-osmolarity medium. KlHog1p phosphorylation via the canonical HOG pathway or in mutants where the SHO1 and SLN1 branches have been inactivated requires not only the presence of KlPbs2p but also its kinase activity. This indicates that when the SHO1 and SLN1 branches are inactivated, high-osmotic-stress conditions activate an independent input that yields active KlPbs2p, which, in turn, renders KlHog1p phosphorylation ineffective. Finally, we found that KlSte11p can alleviate the sensitivity to hyperosmotic stress displayed by a ⌬Klsho1 ⌬Klssk2 mutant when it is anchored to the plasma membrane by adding the KlSho1p transmembrane segments, indicating that this chimeric protein can substitute for KlSho1p and KlSsk2p.

The mating pheromone response pathway in Saccharomyces cerevisiae is one of the best understood signalling pathways in eukaryotes. Comparison of this system with pathways in other fungal species has generated surprises and insights. Cloning and targetted disruption of genes encoding components of the pheromone response pathway has allowed the attribution of specific functions to these signal transduction components. In this review we describe current knowledge of the Kluyveromyces lactis mating system, and compare it with the well-understood S. cerevisiae pathway, emphasizing the similarities and differences in the heterotrimeric G protein activity. This mating pathway is controlled positively by both the Galpha and the Gbeta subunits of the heterotrimeric G protein.

In this work, the effect of the use of two autochthonous starter cultures (Lactobacillus sakei LS131 + Staphylococcus equorum SA25 (EQU), or L. sakei LS131 + Staphylococcus saprophyticus SB12 (SAP)) on the physicochemical, microbiological, proteolytic and lipolytic changes taking place during the manufacture of Galician chorizo, a traditional Spanish sausage, was studied. Three different batches (control (CNT), EQU and SAP) were manufactured in triplicate and analysed during the manufacturing process (samples were taken and analysed at 0, 2, 5, 9, 14, 21 and 30 days of ripening) for proximate composition, pH, aw, colour parameters, nitrogen fractions, free amino acids, biogenic amines, fat parameters and free fatty acids. The use of either of these two starter cultures slightly but significantly reduced the pH values during the fermentation and increased the percentage of transformation to nitrosyl-heme pigments as well as the a* and b* values in the final products. The two starters significantly decreased the Enterobacteriaceae counts in the final product, but without this microbial group completely disappearing. Both starter cultures significantly increased the α-amino acidic nitrogen and the total basic volatile nitrogen fractions during manufacturing, also increasing the free amino acid content and reducing the total biogenic amine content by approximately 20%. The SAP starter enhanced the lipolytic processes, increasing the free fatty acid content. Due to their performances, these two starter cultures seem to be suitable for increasing the quality and safety of the Galician chorizo sausage.

Mating in yeast is initiated by binding of pheromone to G-protein-coupled receptors expressed in haploid cells. We analysed the role of KlSte2p and KlSte3p receptors in the Kluyveromyces lactis mating pathway. By sequence analysis, KlSte2p and KlSte3p are the homologues of the Saccharomyces cerevisiae α-pheromone and a-pheromone receptors, respectively. However, by expression experiments, we determined that KlSTE2 gene is expressed in the cells typified as MAT α and therefore is the receptor for the K. lactis a-pheromone and KlSTE3 gene is expressed in the MAT a cells and binds the α-pheromone. The KlSTE2 gene is silent in MAT a cells, while it is highly expressed in MATα cells, and conversely the KlSTE3 gene is expressed in MAT a cells and repressed in MATα cells. Disruption mutants of both genes were found to be sterile, and this defect is reversed by plasmidic copies of each gene. The cytoplasmic C-terminus of KlSte3p interacts strongly with the KlGpa1p (Gα) subunit, which is involved in the transduction of the pheromone stimulus to induce mating. Remarkably, this same domain does not interact with a constitutive active allele of the Gα subunit, indicating that the C-terminus is able to discriminate between the active (GTP-bound) and inactive (GDP-bound) forms of the Gα subunit.

The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis.

Yeast cells respond to hyperosmotic stress by activating the high-osmolarity glycerol (HOG) pathway, which consists of two branches, Hkr1/Msb2-Sho1 and Sln1, which trigger phosphorylation and nuclear internalization of the Hog1 mitogen-activated protein kinase. In the nucleus, Hog1 regulates gene transcription and cell cycle progression, which allows the cell to respond and adapt to hyperosmotic conditions. This study demonstrates that the uncoupling of the known sensors of both branches of the pathway at the level of Ssk1 and Ste11 impairs cell growth in hyperosmotic medium. However, under these conditions, Hog1 was still phosphorylated and internalized into the nucleus, suggesting the existence of an alternative Hog1 activation mechanism. In the ssk1ste11 mutant, phosphorylated Hog1 failed to associate with chromatin and to activate transcription of canonical hyperosmolarity-responsive genes. Accordingly, Hog1 also failed to induce glycerol production at the levels of a wild-type strain. Inactivation of the Ptp2 phosphatase moderately rescued growth impairment of the ssk1ste11 mutant under hyperosmotic conditions, indicating that downregulation of the HOG pathway only partially explains the phenotypes displayed by the ssk1ste11 mutant. Cell cycle defects were also observed in response to stress when Hog1 was phosphorylated in the ssk1ste11 mutant. Taken together, these observations indicate that Hog1 phosphorylation by noncanonical upstream mechanisms is not sufficient to trigger a protective response to hyperosmotic stress.

The different vegetable oils used in canned fish as a filling medium have a preserving effect and contribute to the palatability of the product. In this study, the colour of European eels and the filling medium (sunflower oil, olive oil or spicy olive oil) was measured at different steps of the canning process. The sensorial characteristics of canned eels packed in the different oils were also evaluated. Colour scores (CieLab values) were higher in canned eels packed in sunflower and spicy olive oil than in canned eels packed in olive oil. The changes in colour parameters depended on the type of oil, the stage of the process and the storage time. Colour changes in canned eels packed in olive oil were highest during the sterilization process. Spicy olive oil was the filling medium in which the colour change was greatest, probably due to the migration of some of the spice components into the oil. Organoleptic properties were directly related to the type of oil used as the filling medium. The canned eels packed in sunflower oil were those awarded the highest scores in consumer tests, although the preferences varied depending on the age and gender of the consumers.

The different vegetable oils used in canned fish as filling medium have a preserving effect and contribute to the palatability of the product. In this study, the colour of European eels and the filling medium (sunflower oil, olive oil or spicy olive oil) was measured at different steps of the canning process. The sensorial characteristics of canned eels packed in the different oils were also evaluated. Colour scores (CieLab values) were higher in canned eels packed in sunflower and spicy olive oil than in canned eels packed in olive oil. The changes in colour parameters depended on the type of oil, the stage of the process and the storage time. Colour changes in canned eels packed in olive oil were highest during the sterilization process. Spicy olive oil was the filling medium in which the colour change was greatest, probably due to the migration of some of the spice components into the oil. Organoleptic properties were directly related to the type of oil used as the filling medium. The canned eels packed in sunflower oil were those awarded the highest scores in consumer tests, although the preferences varied depending on the age and gender of the consumers.