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on line novembre 2017paper : janvier 2018; Yeasts play a crucial role in cheese ripening. They contribute to the curd deacidification, the establishment of acid-sensitive bacterial communities, and flavour compounds production via proteolysis and catabolism of amino acids (AA). Negative yeast-yeast interaction was observed between the yeast Yarrowia lipolytica 1E07 (YL1E07) and the yeast Debaryomyces hansenii 1L25 (DH1L25) in a model cheese but need elucidation. YL1E07 and DH1L25 were cultivated in mono and co-cultures in a liquid synthetic medium (SM) mimicking the cheese environment and the growth inhibition of DH1L25 in the presence of YL1E07 was reproduced. We carried out microbiological, biochemical (lactose, lactate, AA consumption and ammonia production) and transcriptomic analyses by microarray technology to highlight the interaction mechanisms. We showed that the DH1L25 growth inhibition in the presence of YL1E07 was neither due to the ammonia production nor to the nutritional competition for the medium carbon sources between the two yeasts. The transcriptomic study was the key toward the comprehension of yeast-yeast interaction, and revealed that the inhibition of DH1L25 in co-culture is due to a decrease of the mitochondrial respiratory chain functioning.

All organisms need to sense and respond to a range of stress conditions. In this study, we used transcriptional profiling to identify genes and cellular processes that are responsive during arsenite and tert-butyl hydroperoxide exposure in Kluyveromyces lactis. Many arsenite-responsive genes encode proteins involved in redox processes, protein folding and stabilization, and transmembrane transport. The majority of peroxide-responsive genes encode functions related to transcription, translation, redox processes, metabolism and transport. A substantial number of these stress-regulated genes contain binding motifs for the AP-1 like transcription factors KlYap1 and KlYap8. We demonstrate that KlYap8 binds to and regulates gene expression through a 13 base-pair promoter motif, and that KlYap8 provides protection against arsenite, antimonite, cadmium and peroxide toxicity. Direct transport assays show that Klyap8 Delta cells accumulate more arsenic and cadmium than wild type cells and that the Klyap8 Delta mutant is defective in arsenic and cadmium export. KlYap8 regulates gene expression in response to both arsenite and peroxide, and might cooperate with KlYap1 in regulation of specific gene targets. Comparison of KlYap8 with its Saccharomyces cerevisiae orthologue ScYap8 indicates that KlYap8 senses and responds to multiple stress signals whereas ScYap8 is only involved in the response to arsenite and antimonite. Thus, our data suggest that functional specialization of ScYap8 has occurred after the whole genome duplication event. This is the first genome-wide stress response analysis in K. lactis and the first demonstration of KlYap8 function.

The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungi-yeasts split concomitant with the yeasts' genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts.

Sulfur metabolism is a central function of the cell. It has been extensively studied in the model yeast Saccharomyces cerevisiae. A comparative genomic study carried out across the hemiascomycetes clade has shown that S. cerevisiae displayed specificities not shared by the other yeast species. For instance, an O-acetylserine pathway was shown to be present in many yeast species. The complex regulatory pathways seem also to be conserved, with the exception of MET28, whose presence seems to be restricted to S. cerevisiae and related species. In order to explore this pathway in two distant yeast species, Kluyveromyces lactis and Yarrowia lipolytica, transcriptomic and metabolomic studies have been carried out in different conditions of sulfur supply. These high-throughput techniques allowed confirmation of the data of the comparative genomics but also the investigation of new components and new functions linked to sulfur metabolism, for instance, the role of the O-acetylserine pathway in cysteine biogenesis and the role of the aminotransferases in the degradation of methionine were confirmed. The screening of the pools of metabolic intermediates affected by the sulfur supply allowed the identification of new components of the pathway in Y. lipolytica such as taurine and hypotaurine, which seemed to play a role of sulfur storage. These methods also allowed the identification of the set of transporters involved in sulfur metabolism. Eventually, the comparison of these results with the data accumulated in the model S. cerevisiae highlighted the large-scale conservation of this pathway but also the large diversity in the regulated steps inside the pathway.

The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei , Brevibacterium aurantiacum , and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed.

International audience; With the increasing threat of environmental toxicants including biological and chemical warfare agents, fabricating innovative biomimetic systems to detect these harmful agents is critically important. With the broad objective of developing such a biosensor, here we report the construction of a Saccharomyces cerevisiae strain containing the primary components of the mammalian olfactory signaling pathway. In this engineered yeast strain, WIF-1alpha, olfactory receptor signaling is coupled to green fluorescent protein expression. Using this 'olfactory yeast', we screened for olfactory receptors that could report the presence of the odorant 2,4-dinitrotoluene, an explosive residue mimic. With this approach, we have identified the novel rat olfactory receptor Olfr226, which is closely related to the mouse olfactory receptors Olfr2 and MOR226-1, as a 2,4-dinitrotoluene-responsive receptor.

Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.

International audience; Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.

International audience; Extracellular signals are transduced into cells through mitogen-activated protein kinases (MAPKs), which are activated by their upstream kinases. Recently, families of scaffolding proteins have been identified to tether specific combinations of these kinases along specific signaling pathways. Here we describe a protein, JLP (c-Jun NH2-terminal kinase-associated leucine zipper protein), which acts as a scaffolding protein to bring together Max and c-Myc along with JNK (c-Jun NH2-terminal kinase) and p38MAPK, as well as their upstream kinases MKK4 (MAPK kinase 4) and MEKK3 (MAPK kinase kinase 3). Thus, JLP defines a family of scaffolding proteins that bring MAPKs and their target transcription factors together for the execution of specific signaling pathways.

International audience; Expression of the GTPase-deficient, activated mutant alpha-subunit of the heterotrimeric G protein G12 (Galpha12QL) leads to the neoplastic transformation of fibroblast cell lines. The mitogenic pathway regulated by Galpha12QL includes an extensive signaling network involving several small GTPases and various kinases. In addition, Galpha12QL has been shown to potentiate the serum-induced phospholipase-A2 activity in NIH3T3 cells. In the present study, we demonstrate that cycloxygenase-2 (COX-2) pathway is involved in the mitogenic pathway activated by Galpha12QL. Expression of Galpha12QL and not Galpha13QL, stimulates the serum-induced release of arachidonic acid in NIH3T3 cells. Furthermore, expression of Galpha12QL or the stimulation of wild-type Galpha12 induces the expression of COX-2. Our results also indicate that the COX-2 inhibitor acutely disrupts the DNA-synthesis stimulated by Galpha12QL in NIH3T3 cells. These studies, for the first time, identify the crucial role of COX-2 in Galpha12-mediated regulation of cell proliferation and suggest a role for prostaglandin-derived autocrine loop in Galpha12-mediated signaling pathways.

Apoptosis of the ovarian granulosa cells plays a crucial role in the determination of the number of follicles destined to ovulate in each reproductive cycle. While the activation of specific apoptotic pathway or the inactivation of cell survival pathway can initiate apoptosis, the signaling mechanism(s) involved in initiating the onset of apoptosis in granulosa cells is not fully understood. In the present study, using granulosa cells derived from eCG-primed immature rats, we investigated the temporal signaling events involved in the onset of apoptosis in the granulosa cells. The administration of 15 IU of eCG to 21-day-old immature female rats stimulate the growth and development of ovarian follicles until 72 h, after which the granulosa cells of the ovarian follicles undergo apoptosis due to the waning levels of tropic hormonal support. An analysis of the signaling events leading to apoptosis indicates that the DNA fragmentation can be seen in these cells from 96 h. A small increase in the levels of the pro-apoptotic factor Bax can be seen from 96 h while an increase in the activity of JNK can be seen from 108 h onwards. By contrast, a reduction in ERK signaling can be seen by 48 h. Similar reduction in Raf-1 kinase activity can be discerned from 48 h onwards. A concomitant decrease in the phosphorylated form of Bad can also be detected. These findings taken together, suggest that the loss of tropic hormone support is translated into the attenuation of Raf-1-MEK-ERK signaling pathway and this reduction along with a reduction in the levels of phosphorylated form of Bad triggers the onset of apoptosis in the ovarian granulosa cells. J. Cell. Biochem. 75:547–554, 1999. © 1999 Wiley-Liss, Inc.

International audience; G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the alpha-subunits as well as the betagamma-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Of the 17 alpha-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in fibroblast cells. Activating mutations in G alpha(s), G alpha(i)2, and G alpha12 have been correlated with different types of tumors. In addition, the ability of the betagamma-subunits to activate mitogenic pathways in different cell-types has been defined. The present review briefly summarizes the diverse and novel signaling pathways regulated by the alpha- as well as the betagamma-subunits of G proteins in regulating cell proliferation.