Final Report Summary - BACEMO (Bacterial Cell Morphogenesis)
In bacteria, the though external cell wall and the intracellular actin-like (MreB) cytoskeleton are major determinants of cell shape. The biosynthetic pathways and chemical composition of the cell wall, a three dimensional polymer network that is one of the most prominent targets for antibiotics, are well understood. However, despite decades of study, little is known about the complex cell wall ultrastructure and the molecular mechanisms that control cell wall morphogenesis in time and space. In rod-shaped bacteria, actin-like MreB homologues assemble into dynamic structures thought to control shape by serving as organizers for the movement and assembly of macromolecular machineries that effect sidewall elongation. However, the mechanistic details used by the MreB cytoskeleton to fulfil this role remain to be elucidated. Furthermore, although the ability of MreB proteins to form polymers was well established, the structure, length, and conditions of establishment of MreB polymers in vivo was the subject of conflicting reports and remained unclear. Elucidating the true structure of MreB assemblies was nevertheless important because it determines the models describing how cell wall synthesizing machineries are associated, how they are coordinated at the cellular scale, and how MreB mediates this regulation.
This project combined powerful genetic, biochemical, genomic and systems biology approaches with modern high-resolution fluorescence microscopy techniques to study the dynamics, ultrastructure and morphogenetic role of MreB assemblies in rod-shaped bacteria. We investigated the dynamics of MreB assemblies, as a proxy for cell synthesis, during growth and in response to nutrient-dependent growth rate changes in the model Gram-positive bacterium Bacillus subtilis and in the model Gram-negative Escherchia coli. We showed these two model organisms use orthogonal strategies to achieve different growth rates and cell sizes, and developed numerical and schematic models that link MreB-mediated cell wall synthesis and cell elongation. We also showed that in B. subtilis MreB forms sub-diffraction (< 200 nm-long) nanofilaments during active growth, arguing against previous models in which MreB long-range filaments would coordinate distant machineries along the sidewall. Besides, our functional studies in B. subtilis connected the MreB cytoskeleton with peptidoglycan hydrolysis, genetic competence, oxidative stress and second messengers used in signal transduction in bacteria. Furthermore, we screened a genomic knock-out library of B. subtilis to identify mutants involved in the acquisition and maintenance of cell diameter, and developed a reporter system for the screening of anti-MreB antimicrobials. Finally, using single-cell analysis, we were instrumental in the study of the localization, dynamics and role of actin-like MreB and tubulin-like FtsZ in the intracellular pathogen Shigella.
In summary, this project used complementary, integrative cutting-edge imaging and systems biology approaches to bring new insights into the bacterial actin cytoskeleton and bacterial cell wall morphogenesis. We generated and integrated knowledge of protein localization and dynamics, interaction partners, and genetic and physical factors underlying cell wall biosynthesis as well as their relationship with the MreB cytoskeleton. Our research opened new perspectives for the understanding of both bacterial cell architecture and organization and cytoskeletal processes in living cells.
This project combined powerful genetic, biochemical, genomic and systems biology approaches with modern high-resolution fluorescence microscopy techniques to study the dynamics, ultrastructure and morphogenetic role of MreB assemblies in rod-shaped bacteria. We investigated the dynamics of MreB assemblies, as a proxy for cell synthesis, during growth and in response to nutrient-dependent growth rate changes in the model Gram-positive bacterium Bacillus subtilis and in the model Gram-negative Escherchia coli. We showed these two model organisms use orthogonal strategies to achieve different growth rates and cell sizes, and developed numerical and schematic models that link MreB-mediated cell wall synthesis and cell elongation. We also showed that in B. subtilis MreB forms sub-diffraction (< 200 nm-long) nanofilaments during active growth, arguing against previous models in which MreB long-range filaments would coordinate distant machineries along the sidewall. Besides, our functional studies in B. subtilis connected the MreB cytoskeleton with peptidoglycan hydrolysis, genetic competence, oxidative stress and second messengers used in signal transduction in bacteria. Furthermore, we screened a genomic knock-out library of B. subtilis to identify mutants involved in the acquisition and maintenance of cell diameter, and developed a reporter system for the screening of anti-MreB antimicrobials. Finally, using single-cell analysis, we were instrumental in the study of the localization, dynamics and role of actin-like MreB and tubulin-like FtsZ in the intracellular pathogen Shigella.
In summary, this project used complementary, integrative cutting-edge imaging and systems biology approaches to bring new insights into the bacterial actin cytoskeleton and bacterial cell wall morphogenesis. We generated and integrated knowledge of protein localization and dynamics, interaction partners, and genetic and physical factors underlying cell wall biosynthesis as well as their relationship with the MreB cytoskeleton. Our research opened new perspectives for the understanding of both bacterial cell architecture and organization and cytoskeletal processes in living cells.