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Etude et caractérisation de l'état " Viable mais Non Cultivable " chez Saccharomyces cerevisiae

Abstract : The viable but not culturable (VBNC) state has been studied in detail in bacteria. Bycontrast the VBNC state in other microorganisms, including particularly eukaryotes, has receivedmuch less attention. However, it has been suggested that in wine, Brettanomyces yeast cells mayenter a Viable But Not Culturable State, in particular in the presence of high, sulfur dioxide(SO2) concentration.To provide conclusive evidences for the existence of a VBNC state in yeast, especially in S.cerevisiae as a model organism, the capacity of different S cerevisiae strains to become viableand not cultivable after a sulfite stress with various concentrations of SO2 was studied by flowcytometry (FCM) using fluorescent probe as a viability marker (Fluorescein diacetate (FDA))and by plating on culture medium. The ability of cells to recover cultivability after stress removalby increasing the pH medium was investigated. To confirm the VBNC state, the rate ofgeneration of VBNC cells after stress removal was compared to cultivable and viable cells insame culture conditions. In addition, the comparison of different cell cycle phases of cells exitingthe VBNC state and cells in VBNC state was performed by FMC. Moreover, the involvement ofSSU1 gene coding for the SO2 pump in VBNC state was studied.After stress application, comparison between cultivable population determined on culturemedium and viable population assessed by FCM demonstrated the presence of the viable cellswhich became uncultivable after 24 to 48 hours depending on the strains under study. Increasingthe pH medium allows the viable but uncultivable S. cerevisiae cells to become cultivable again.The generation rate of cells exiting VBNC state was not consistent with growth of residualculturable cells, which support a true VBNC state. The absence of cell proliferation, the stabilityof the population during the increase of the cultivability and the decrease in esterase activity forVBNC cells allows us to conclude the presence of the VBNC state in S. cerevisiae in correlationwith the VBNC state definition.In order to determine whether SSU1 gene, encoding a sulfite pump efflux, was involved inVBNC, we compare a wild type S. cerevisiae strain to its nul mutant Δ ssu1. Our resultsdemonstrate that SSU1 gene does not seem to be involved in VBNC phenotype
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Mohammad Salma. Etude et caractérisation de l'état " Viable mais Non Cultivable " chez Saccharomyces cerevisiae. Sciences agricoles. Université de Bourgogne, 2013. Français. ⟨NNT : 2013DIJOS041⟩. ⟨tel-00973496⟩

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