Investigation of MreB dynamics and cell wall synthesis in B. subtilis using superresolution microscopy and optical-mechanical manipulation techniques

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Understanding the structure and synthesis of the cell wall of bacteria is of great importance in fundamental research, but also crucial for the development of new drugs, especially antibiotics. For these reasons, during the last years several groups have focused their research on the actin-like protein MreB, which is part of the bacterial cytoskeleton and plays a crucial role in cell wall synthesis. In a collaboration with the lab of Prof. Graumann (University of Marburg) and using fast superresolution fluorescence microscopy (TIRF-SIM) as well as biophysical analysis we could show that MreB filaments exhibit a length-dependent velocity. Furthermore, these filaments undergo frequent changes of transport direction and seem to serve as a mechanical coupler during the synthesis of peptidoglycan strands in the cell wall. These observations were made in bacteria of the type Bacillus subtilis, which will serve as a model system for the proposed investigations.In this project an existing superresolution microscope that combines total internal reflection with structured illumination shall be extended by additional features and significantly improved in terms of acquisition speed. Using this set-up we want to observe the dynamics of MreB and other cell wall-related proteins with an optical resolution of almost 100nm. Furthermore, we want to implement a second visible laser for fluorescence excitation. This will enable the observation of MreB and MreB-interacting enzymes of the cell wall synthetic machinery in colocalization experiments. Additionally, we want to implement an IR-laser for optical tweezing and a UV-laser for micro-dissection.According to our recently presented, mechanistic model, MreB organzises the synthesis of several cell wall strands. This is to be tested experimentally under more complex conditions. These results shall be used to extend our mathematical model, which is based on coupled molecular motors, with the aim to describe all observed phenomena in MreB dynamics and cell wall synthesis. Comparisons between theory and experiment will improve the qualitative and quantitative understanding of cell wall synthesis.We also want to investigate how global and local perturbations of the bacteria influence cell wall synthesis. For a global manipulation all bacteria of one sample can be treated with a defined amount of biochemical reactant. The consequences should become apparent by observing MreB and other relevant proteins by fluorescence microscopy. In a second step, we want to perturb single bacteria locally by the means of optical and mechanical forces which can lead to local changes in protein dynamics. These manipulations should be done either by optical tweezers or by the use of a UV-laser that can locally destroy cell material and deactivate proteins. Hereby, we want to address the question if cell wall synthesis and MreB dynamics are locally self-regulated or rather globally controlled by the cell.