ED Sciences de la Vie et de la Santé
Morphogenesis of Mycoplasma capricolum subsp. capricolum induced by MreB1 from Spiroplasma citri: cellular localization and functional determinants of the protein
by Julien PEROCHON (BFP - Biologie du Fruit et Pathologie)
The defense will take place at 14h00 - Amphithéâtre Colette et Josy Bové 71, avenue Edouard Bourlaux, 33882 Villenave d'Ornon cedex Amphithéâtre Colette et Josy Bové
in front of the jury composed of
- Laure BEVEN - Professeure des universités - Université de Bordeaux - Directeur de these
- Eric BARANOWSKI - Directeur de recherche - INRAE, ENVT - Rapporteur
- Arnaud CHASTANET - Chargé de recherche - INRAE, Micalis - Rapporteur
- Nienke BUDDELMEIJER - Chargée de recherche - Institut Pasteur - Examinateur
- Sabine PEREYRE - Professeure des universités - praticienne hospitalière - Université de Bordeaux - Examinateur
- Christophe GRANGEASSE - Directeur de recherche - Université Claude Bernard Lyon - Examinateur
Spiroplasmas are polarized, wall-less helical bacteria that possess a naturally minimal molecular system for locomotion. In these organisms, helicity and motility are tightly coupled: a curvature initiated at the anterior pole propagates along the cell body, inducing a change in helicity that enables swimming in semi-viscous fluids. Actin-like ATPases (SMreBs), together with the fibril protein, form a cytoskeletal structure responsible for the helical morphology and motility of these bacteria. The lack of efficient genetic tools in spiroplasmas (such as our model Spiroplasma citri) led us to express different SMreB isoforms in Mycoplasma capricolum subsp. capricolum (Mcap) in order to investigate the respective roles of these cytoskeletal proteins. Mcap is a pleomorphic bacterium phylogenetically related to spiroplasmas and lacking actin-like proteins. Expression of the single ScMreB1 is sufficient to induce cellular helicity, whereas ScMreB5 induces both helicity and the propagation of membrane deformations mimicking the spiroplasmic motility mechanism. This suggests a functional diversity among MreB proteins, with some acting as structural components, such as MreB1, and others functioning as motility-associated proteins, such as MreB5. In this thesis work, the assessment of the impact of MreB1 expression level on Mcap morphology suggests the existence of a phase transition between a monomeric state and a membrane-coupled mechanically polymerized state, governed by a threshold in the expression level and effective density of MreB1. The use of expansion microscopy enabled us to visualize the cellular localization of the protein. Our data suggest that morphogenesis driven by the assembly of MreB1 and its interaction with the cytoplasmic side of the membrane is accompanied by a reorganization of membrane surface topology. We also investigated, through targeted mutagenesis, the intramolecular determinants of MreB1 required for helicity in Mcap. Our findings demonstrate that the N-terminal region is essential for cell elongation and the establishment of a helical cell shape. Moreover, residue D15 appears to be a key determinant of the morphological transition triggered by MreB1 expression, presumably through its role in stabilizing Mg²⁺ and K⁺ ions within the catalytic pocket. Our results also underscore the importance of the pre-hydrolytic MreB1-ATP state for membrane association and/or filament assembly. These studies were further complemented by the development of an atomic force microscopy (AFM) imaging protocol for spiroplasmas. In the long term, this methodology should provide a means to quantify membrane stiffness in both spiroplasmas and mycoplasmas expressing MreB1 at the single-cell level.