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Intégration des bactéries planctoniques dans le biofilm et étude fonctionnelle du gène plasmidique Bthur62720 chez Bacillus thuringiensis

Abstract : Bacillus thuringiensis is able to produce a pellicle at the air-liquid interface in glass tubes under static conditions. During biofilm formation, two populations coexist: a sessile floating population and a planktonic population, located in the culture medium beneath the pellicle. Using spectrophotometric measurements, we followed the growth of both populations during the B. thuringiensis 407 pellicle formation. Our results show that while the biofilm biomass increases rapidly, the planktonic population growth drops sharply. This decrease is not observed with the 407 spoOA mutant or for strains unable to form a biofilm, and cannot be attributed to cell lysis or cell sedimentation. Therefore, it is the result of a massive integration of planktonic cells in the preformed pellicle. We also visualized, using epi-fluorescence microscopy, the integration of planktonic bacteria of the 407 strain in its preformed biofilm. The recruited cells are located in restricted areas of the biofilm, where the density of sessile cells is low, revealing a heterogeneous spatial distribution of the immigrant cells within the biofilm. To identify the mechanisms involved in the recruitment of planktonic cells in the biofilm, we screened a bank of mutants of the 407 strain, obtained by random mutagenesis, for their ability to integrate a pre-existing biofilm. One of the mutants in the library is strongly affected in its ability to integrate a biofilm. This deficiency is caused by the disruption of the Bthur62720 gene, which is carried by the BTB-9p plasmid and encodes a 21 kDa protein. This protein has no homolog and in silico analysis predict a signal peptide, a N-terminal domain of unknown function and a C-terminal membrane domain. Using immunocytochemistry and translational fusion assays with GFP, we showed that this protein is parietal, polar and that its N-terminal domain is cytoplasmic. With a specific dye of charged membrane phospholipids, 10-N-nonyl acridine orange, we showed that the deletion of Bthur002_62720 disorganizes the lipid rafts distribution, which appear essentially polar in wild type strain 407. Moreover, this deletion strongly affects linear swimming, but not bacterial tumbling or the presence of flagella. These results allow us to hypothesis that Bthur62720 stabilizes the lipid rafts located at the cell poles. The polar localization of these rafts, required for the clustering of chemoreceptors, would be necessary to ensure a normal chemotaxis function and thus, bacterial swimming.
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Submitted on : Friday, April 16, 2021 - 6:45:22 PM
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Nay El Khoury. Intégration des bactéries planctoniques dans le biofilm et étude fonctionnelle du gène plasmidique Bthur62720 chez Bacillus thuringiensis. Bactériologie. Université Paris-Saclay; Université Saint-Joseph (Beyrouth), 2021. Français. ⟨NNT : 2021UPASL014⟩. ⟨tel-03200788⟩

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