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Characterization of the Actin-Like Mreb Cytoskeleton Dynamics and Its Role in Cell Wall Synthesis in Bacillus Subtilis Julia Domínguez Escobar

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Characterization of the Actin-Like Mreb Cytoskeleton Dynamics and Its Role in Cell Wall Synthesis in Bacillus Subtilis Julia Domínguez Escobar Characterization of the actin-like MreB cytoskeleton dynamics and its role in cell wall synthesis in Bacillus subtilis Julia Domínguez Escobar Dissertation and der Fakultät für Biologie der Ludwig-Maximilians-Universität München Dissertation zur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität München Characterization of the actin-like MreB cytoskeleton dynamics and its role in cell wall synthesis in Bacillus subtilis vorgelegt von Julia Domínguez-Escobar aus Mexiko München, den 13. Februar 2013 Erstgutachter: Prof. Dr. Thorsten Mascher Zweitgutachter: Prof. Dr. Marc Bramkamp Tag der mündlichen Prüfung: 13.05.2013 Abstract The peptidoglycan cell wall (CW) and the actin-like MreB cytoskeleton are the major determinants of cell morphology in non-spherical bacteria. Bacillus subtilis is a rod-shaped Gram- positive bacterium that has three MreB isoforms: MreB, Mbl (MreB–like) and MreBH (MreB- Homologue). Over the last decade, all three proteins were reported to localize in dynamic filamentous helical structures running the length of the cells underneath the membrane. This helical pattern led to a model where the extended MreB structures act as scaffolds to position CW-synthesizing machineries along sidewalls. However, the dynamic relationship between the MreB cytoskeleton and CW elongation complexes remained to be elucidated. Here we describe the characterization of the dynamics of the three MreB isoforms, CW synthesis and elongation complexes in live Bacillus subtilis cells at high spatial and temporal resolution. Using total internal reflection fluorescence microscopy (TIRFM) we found that MreB, Mbl and MreBH actually do not assemble into an extended helical structure but instead into discrete patches that move processively along peripheral tracks perpendicular to the long axis of the cell. We found similar patch localization and dynamics for several morphogenetic factors and CW-synthesizing enzymes including MreD, MreC, RodA, PbpH and PBP2a. Furthermore, using fluorescent recovery after photobleaching (FRAP), we showed that treadmilling of MreB filaments does not drive patch motility, as expected from the structural homology to actin. Blocking CW synthesis with antibiotics that target different steps of the peptidoglycan biosynthetic pathway stopped MreB patches motion, suggesting that CW synthesis is the driving force of patch motility. On the basis of these findings, we proposed a new model for MreB fuction in which MreB polymers restrict and orient patch motility to ensure controlled lateral CW expansion, thereby maintaining cell shape. To further investigate the molecular mechanism underlying MreB action, we next performed a site-directed mutagenesis analysis. Alanine substitutions of three charged amino 1 acids of MreB generated a B. subtilis strain with cell shape and growth defects. TIRFM analysis revealed that the mutated MreB protein displayed wild-type localization and dynamics, suggesting that it is still associated to the CW elongation machinery but might be defective in an interaction important for MreB morphogenetic function. Thus, this mutant appears as as a good candidate to start characterizing the interactions between the three MreB isoforms and components involved in CW elongation. It might also help to understand the function of components of theCW- synthetic complexes, and how they are coordinated to achieve efficient CW synthesis. Finally, to investigate how the integrity of the CW is maintained, we studied the localization and dynamics of the LiaIH-system, which is the target of LiaRS, a two-component system involved in cell envelope stress response. We found that under stress conditions, when liaI and LiaH genes are expressed, the proteins form static complexes that coat the cell membrane. LiaI is required for the even distribution of the LiaH in the membrane. Taken together, these data suggest that LiaIH complexes may protect the cell from CW damage. Taken together, the findings described in this thesis provide valuable insights into the understanding of CW synthesis in B. subtilis, which may open new perspectives for the design of novel antimicrobial agents. 2 Publications 1. Dominguez-Escobar, J., Arnaud Chastanet, A., Crevenna, A. H., Vincent Fromion, V., Wedlich-Söldner, R., and Carballido-López R. (2011) Processive Movement of MreB- Associated Cell Wall Biosynthetic Complexes in Bacteria. Science 333:225-228. 2. Spira, F., Dominguez-Escobar, J., Müller, N., and Wedlich-Söldner, R. (2012) Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy. Journal of Visualized Experiments 63:e3982. 3. Rueff, A.S., Chastanet, A., Domínguez-Escobar, J., Jao, Z., Yates, J., Prejean, M.V., Delumeau, O., Noirot, P., Roland Wedlich-Söldner, R., Filipe, S. R., and Carballido-López, R. (in press) An early cytoplasmatic step of peptidoglycan synthesis is associated to MreB in Bacillus subtlis. Molecular Microbiology. 4. Domínguez-Escobar, J*., Wolf, D*., Fritz, G., Höfler, C., Wedlich-Söldner, R., and Mascher, T. (2013) Localization, Interactions and Cellular Dynamics of the Cell Envelope Stress Proteins LiaI and LiaH in Bacillus subtilis. Manuscript submitted for publication. *Equal contribution. 3 Abbreviations aa amino acid ATP adenosine triphosphate CW cell wall EDTA ethlylene diaminetetraacetic acid F-actin filamentous actin GFP green fluorescent protein GTP guanosine triphosphate Hsp heat shock protein IFs intermedia filaments LPS lipopolysaccharide LTA lipoteichoic acid OD600nm optical density measured at a wavelength of 600 nm ON over night PBP penicillin binding protein PG peptidoglycan RT room temperature TCS two component system TIRFM total internal reflection fluorescence microscopy ts temperature sensitive WACA Walker A cytoskeletal ATPases wt wild-type WTA Wall teichoic acid 4 Contents Abstract ................................................................................................................................................................................. 1 Publications .......................................................................................................................................................................... 3 Abbreviations ....................................................................................................................................................................... 4 Contents ................................................................................................................................................................................ 5 List of figures ........................................................................................................................................................................ 8 List of appendices .............................................................................................................................................................. 10 1. Introduction ..................................................................................................................................................................... 1 1.1. Bacterial cell wall ........................................................................................................................................................... 1 1.1.1. Cell wall architecture .......................................................................................................................................... 2 1.1.2. Peptidoglycan synthesis ...................................................................................................................................... 4 1.1.2.1. Bacillus subtilis PBPs and autolysins .................................................................................................... 7 1.1.2.2. Cell wall synthesis in Bacillus subtilis ................................................................................................... 9 1.2. Bacterial cytoskeleton .......................................................................................................................................... 10 1.2.1. Actin homologues ....................................................................................................................................... 11 1.2.2. Tubulin homologues ......................................................................................................................................... 13 1.2.3. Intermediate filaments homologues................................................................................................................ 14 1.2.4. Additional bacterial cytoskeletal proteins ...................................................................................................... 15 1.3. MreB proteins .............................................................................................................................................................. 16 1.3.1. Biochemical properties of MreB ...................................................................................................................... 17 1.3.2. Subcellular localization of MreB proteins ...................................................................................................... 18 1.3.3. Role of MreB proteins in cell shape determination in Bacilus subtilis ....................................................... 20 1.3.4. Role of MreB proteins in cell wall synthesis in Bacilus subtilis ..................................................................
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