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Resume Les genes recA et sfiA d' Escherichia coli sont des operons SOS regules negativement par le represseur LexA. A l'etat non induit, le taux de synthese de recA est 10 fois plus fort que celui de sfiA si l'on compare le niveau d'expression des fusions d'operon recA::lac et sfiA::lac . Pour etudier les bases moleculaires de cette difference, nous avons compare l'expression de ces deux operons dans des souches ou la concentration du represseur LexA est normale ( lexA + ), nulle (mutation spr ambre) ou plus forte que la normale (en presence du plasmide pJL45, qui porte le gene lexA + lie au promoteur de lac ). Les resultats indiquent (i) que le promoteur de recA est quatre fois plus fort que celui de sfiA (ceci etant mesure dans les bacteries spr ), (ii) qu'aucun des deux operons ne possede un taux d'expression lexA independant qui soit physiologiquement significatif (souches pJL45), et (iii) que l'operateur de recA possede 2,5 fois moins d'affinite que celui de sfiA pour le represseur LexA. Si l'on considere nos resultats precedents qui ont montre que l'operon sfiA (dont l'operateur possede une forte affinite pour LexA) est induit tres rapidement apres le traitement inducteur, et que l'operon recA (dont l'operateur possede une faible affinite pour LexA) est reprime tres rapidement apres l'arret du traitement inducteur, nous concluons que les differences d'affinite des operateurs pour le represseur LexA, n'affectent pas l'inductibilite des operons SOS, mais sert uniquement a regler le niveau de base des differentes fonctions SOS.

A recA::lac operon fusion was constructed using the phage Mu d(Ap, lac) in Escherichia coli to obtain precise measurements of the level of recA gene expression in various genetic backgrounds. The RecA protein normally represents 0.02% of total protein. This value is known to increase dramatically after treatments interrupting DNA synthesis; kinetic experiments showed that the rate of recA expression increases 17-fold within 10 min after UV irradiation or thymine starvation. In mutants affected in SOS regulation or repair the following observations were made: (i) the tif-1 mutation in the recA gene does not alter the basal level of recA expression, suggesting that it improves the protease activity of RecA; (ii) the lexA3 mutation does not create a "super-repressor" of recA; (iii) the tsl-1 mutation in the lexA gene makes the LexA protein a poor repressor of recA at 30 degrees C (2.5-fold derepression) and a poor substrate for RecA protease (3-fold stimulation of recA expression by UV); (iv) the spr-55 amber mutation in the lexA gene causes a 30-fold increase in recA expression, higher than all inducing treatments, and this level cannot be further increased by nalidixic acid; (v) the zab-53 mutation at the recA locus, known to abolish tsl-mediated induction of recA expression, is trans-recessive and thus probably affects a regulatory site on the DNA; (vi) uvrA, B and C, recB and recF mutations do not increase the basal level of recA expression, suggesting that there are not sufficient spontaneous lesions to cause induction even when any one of these three repair pathways is inoperative.

International audience

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Food fermentation and biopreservation processes involve the use of various species and strains of bacteria and yeast. These strains are responsible for the targeted qualities of the food products that are sanitary, organoleptic (aroma and texture) and healthy qualities. The Florilege database project aims at (i) gathering bacterial and yeast phenotypes of food product of interest that are automatically extracted from PubMed-referenced full-text litterature by using a text mining approach (ii) managing the information in a relational database (iii) enabling multi-criteria requests via a Web user-friendly interface. To date 368 phenotypes, 260 synthetised or degraded molecules, 1076 medium or food products, 1181 bacterial taxons have been acquired by a combinaison of automatic and manual annotations of text, used for training the text-mining method.Food products are automatically categorized in Florilege according to the OntoBiotope ontology that we have extended with dairy and bakery products definitions. Taxa are categorized by the NCBI taxonomy. An ontology of microbial characteristics has been specifically enriched by the Florilege project. This ontology defines microbial phenotypes (Ontobiotope-Phenotype), including intracellular characteristics of cells (such as shape, antibiotic resistance...) and microbial uses (Ontobiotope-Use) that express the microbial alteration of the external environment, food or matrix, such as aroma, vitamin or other molecule production, degradation or food coloring.A preliminary Web interface is available for querying taxa, culture medium and food products at http://genome.jouy.inra.fr/Florilege/. The public availability of Florilege database is planned for the end of 2017 with a user-friendly interface for multi-criteria requests and access to various phenotypes.Florilege will be a highly valuable tool to (i) assess phenotypic biodiversity of food microbes (ii) assign biochemical functions to each strain/species from fermented or biopreserved food products (iii) help into the development of innovative food products in particular those that involve fermentation or biopreservation processes.

International audience

International audience

Geotrichum candidum is a hemiascomycetous yeast frequently found in the environment and foodstuffs. It is one of the main yeasts in cheese and it is widely used as adjunct culture in the maturation of cheese. Within ANR project ALIA Food Microbiomes in partnership with industry, we characterized the species the species G. candidum by a multigene phylogenetic study. MLST analysis allowed us to separate the studied strains into two groups. The first contains mainly environmental strains while the second contains only strains isolated from cheese. This suggests a specialization or a selection of a group of strains within industry. We developed a typing method by inter LTR profiles, which can provide a robust tool for an industrial monitoring of strains. The genome of G. candidum CLIB 918 = ATCC 204307 was sequenced. Preliminary analyses revealed evolutionary discontinuities among genes. 6802 genes where identified in which 315 genes have orthologs in filamentous fungi and not in yeast. This suggests that during evolution, G. candidum has retained a large number of genes which have been lost in other yeasts or has received some by horizontal gene transfer. The existence of this other yeasts also having a basal position in hemiascomycetous tree suggests that G. candidum and these other yeasts have an intermediate position during the evolutionary transition fungus to yeast. It is noteworthy that some of them are involved in the metabolism and may play a role in the adaptation of the yeast to the cheese environment.; Geotrichum candidum est une levure hémiascomycète ubiquitaire longtemps considérée comme un champignon filamenteux. C’est l’une des levures les plus fréquemment trouvées dans les fromages dans les quelles elle contribue à l’affinage. Dans le cadre du projet ANR ALIA Food Microbiomes en partenariat avec des industriels fromagers et producteur de levain, nous avons caractérisé l’espèce G. candidum par une étude phylogénétique et placé de manière non ambigüe G. candidum parmi les levures hémiascomètes. Une analyse MLST a permis de séparer les souches étudiées en deux groupes. Le premier contient essentiellement des souches environnementales tandis que le second ne contient que des souches isolé du fromage. Cela suggère une certaine sélection ou spécialisation d’un groupe de souche dans la fabrication du fromage. Une méthode de typage inter LTR plus discriminante a permis de typer l’ensemble des souches et peut fournir aux industriels un outil robuste pour le suivi d’une souche en production. Le génome de G. candidum CLIB 918 = ATCC 204307 a été séquencé. Les premières analyses ont mis en évidence des discontinuités évolutives parmi les gènes qui le composent. Parmi les 6802 gènes identifiés, 315 gènes présentent des orthologues chez les champignons filamenteux et non chez les levures. Cela suggère que durant l’évolution, G. candidum a conservé un grand nombre de gènes qui a été perdu chez les autres levures ou en a reçu certain par transfert horizontal de gènes. L’existence de ce même type de gènes chez d’autres levures ayant une position basale dans l’arbre des hémiascomycètes, suggère que G. candidum et ces levures ont une position intermédiaire lors de la transition évolutive champignon vers levure. Il est à noter que certains d’entre eux sont impliqués dans le métabolisme et pourraient jouer un rôle dans l’adaptation de cette levure à la fabrication du fromage.

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.

Diplôme : Dr. d'Universite; Kluyveromyces lactis is one of the most important yeast in cheeses. It is of double interest: fundamental and industrial. Indeed, in spite of its close phylogenetic proximity to the classical model Saccharomyces cerevisiae, its physiology is very different. Its weaker fermentative activity makes of it a model of choice for recombinant proteins production and its ability to metabolize the lactose justifies its utilization for dairy industry. Although K. lactis is the most studied non-conventional yeast, many aspects of its physiology remained to be understood, what we undertook thanks to the data of the partial sequencing of its genome. This work started with the construction of a DNA microarray representing 482 K. lactis genes, involved mainly in the main cellular functions. The comparison of gene expression between the reference strain and an industrial strain in the presence of glucose and/or lactose evidenced a variability that was not detectable at the molecular level. We observed in particular changes in the expression of the genes involved in glucose and lactose transport, in the glycolysis, the Pentose Phosphate Pathway, the Tricarboxylic Acids Cycle, the ethanol metabolism and the amino-acids metabolism, which were perfectly correlated with the physiological data made in parallel. Our results show a very different sensitivity to the glucose repression between both strains, which may be linked to a difference in the sugar transport and may reflect the way by which K. lactis adapts to its environment.; Kluyveromyces lactis est l'une des espèces de levures majoritaires dans les fromages. Elle présente un intérêt à la fois fondamental et industriel. En effet, malgré sa proximité relative du modèle classique Saccharomyces cerevisiae sa physiologie est très différente. Sa plus faible activité fermentaire en fait un organisme de choix pour la production de protéines recombinantes et sa capacité à métaboliser le lactose justifie son utilisation en industrie laitière. Bien que K. lactis soit la levure non conventionnelle la plus étudiée, de nombreux aspects de sa physiologie restaient à éclaircir, ce que nous avons entrepris grâce aux données du séquençage partiel de son génome. Ce travail a débuté par l'élaboration d'une puce à ADN représentant 482 gènes de K. lactis, impliqués dans les principales fonctions cellulaires. La comparaison de l'expression des gènes entre la souche de référence et une souche industrielle en présence de glucose et/ou de lactose a mis en évidence une variabilité indétectable du point de vue moléculaire. Nous avons observé en particulier des changements d'expression des gènes impliqués dans le transport du glucose et du lactose, la glycolyse, la voie des pentoses phosphate, le cycle de Krebs, le métabolisme de l'éthanol et le métabolisme des acides aminés, parfaitement corrélées aux données physiologiques effectuées en parallèle. Nos résultats montrent notamment une sensibilité à la répression par le glucose totalement différente entre les deux souches, qui pourrait être liée à une différence de transport des sucres et refléter la façon dont K. lactis s'adapte à son environnement.