6533b7d3fe1ef96bd1261093

RESEARCH PRODUCT

Modification de la composition lipidique membranaire chez les bactéries lactiques en conditions de stress : étude du rôle physiologique des Acides Gras Cycliques chez deux modèles : oenococcus oeni ATCC-BAA1163 et Lactococcus lactis MG1363

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The results obtained in this work have shown that in both lactic acid bacteria models, L. lactis subsp. cremoris and O. oeni, transcription of the cfa gene, encoding the Cfa synthase is stimulated when cells enter stationary phase of growth or when the cells are grown under stress conditions (acidic or presence of ethanol in the medium). The role of the CFAs has been apprehended by the comparative physiological analysis of the parental strain L. lactis subsp. cremoris and a Δcfa mutant generated in this strain. The high percentages of survival obtained in strains grown at pH 5.0 and undergoing acid stress (pH 3.0) prove that the cyclopropanation of unsaturated fatty acids is not essential to the survival of L. lactis subsp. cremoris under conditions of acid stress. In addition, fluorescence anisotropy data show that the presence of membrane CFA does not regulate the level of membrane fluidity, especially with strains grown in the presence of ethanol. The assumption of a balance between palmitoleic acid / cis-vaccenic acid / lactobacillic acid in membrane fluidity regulation in L. lactis subsp. cremoris is advanced. The study of the physiological role of CFA in O. oeni requires cfa gene expression of the bacterium in a heterologous system. The results confirm the functionality of the gene in the host bacterium L. lactis subsp. cremoris. However, the cyclopropanation of the precursor cis-vaccenic acid remains partial in the mutant strain complemented. Biochemical characterization in vitro of the enzyme CFA synthase from O. oeni was therefore undertaken in order to explain the low efficiency of the enzyme of O. oeni in the host bacterium. The work led to the overproduction and purification of the enzyme. A technique for measuring the activity in vitro has also been developed. A strategy of O. oeni cfa gene expression in the heterologous host bacterium E. coli BL21 Star has permitted the surpoduction of a protein of approximately 45 kDa. This data is in agreement with the theoretical molecular weight of the CFA synthase from O. oeni. Protein purification was performed by affinity chromatography on nickel-agarose column. Enzymatic activity assays in vitro have been made. The optimum temperature for the enzyme is 35.8 ° C. Its optimum pH is 5.6. However, we find that the in vitro cyclopropanation of cis-vaccenic acid from membrane phospholipids of the mutant L. lactis subsp. cremoris Δcfa remains partial. This leads to an experimental determination of KM and Vmax values much higher than the data cited in the literature. The difference between the nature and the distribution of membrane phospholipids in the two model bacteria may explain the partial activity of Cfa synthase from O. oeni.