Molecular view on the spatiotemporal organization of Bacillus subtilis subcellular compartments

All living cells are highly organized and exhibit complex cellular machineries facilitating biochemical reactions. Compartmentalization is a prerequisite to allocate an appropriate environment for these processes. In this work, compartments that are involved in Bacillus subtilis membrane organization and cell division were studied. B. subtilis division site selection is dependent on the nucleoid occlusion and the Min system. The B. subtilis Min system consists of four components. MinC is the actual inhibitor of the tubulin homologue FtsZ that is a crucial component of the divisome, forming the so called Z-ring. MinC is bound to the ATPase MinD that is tethered via the adapter protein MinJ to DivIVA. DivIVA senses membrane curvature and was supposed to be stably tethered to the cell poles. Thereby a stable, static DivIVA / MinJDC gradient with minimum concentration at midcell is formed. Using advanced microscopy techniques like single cell time lapse microscopy, fluorescence recovery after photo bleaching and by utilization of photo-activatable / convertible fluorophores we could demonstrate that DivIVA is in vegetative cells recruited from the cell pole to mature septa. These data provide first evidence that the role of the B. subtilis Min system is not to define midcell, but prevents reinitiation of Z-ring constriction after fulfilled division. Utilizing single cell time lapse microscopy we could further demonstrate that proteins crucial to condense the chromosome are vital for correct chromosome segregation during cell division by influencing the replication fork velocity or resolution. As a second compartment B. subtilis flotillin dependent membrane microdomains were studied. These domains are likely scaffolded by the membrane protein flotillin. This protein is pinned to the membrane via a hairpin loop as shown by SNAP–tag labelling experiments. Utilizing the anisotropic dye Laurdan we could further show spectroscopically and microscopically that flotillins prevent condensation of microdomains. Flotillin deletion strains also exhibit a generally more liquid ordered membrane compared to wild type cells. Using co–immunoprecipitation experiments several proteins interacting with flotillin were identified. These interactions were confirmed with microscopical co–localization analysis. B. subtilis flotillin was additionally heterologously purified via affinity chromatography. The purified protein creates large homo–oligomers likely in mega Dalton size. Using truncation mutants it could be shown that flotillin oligomerizes via a flotillin specific domain, namely the PHB domain. Though, contrary to eukaryotic cells, B. subtilis PHB domain does not contribute to lipid binding. However, several cellular machineries that interact with flotillins, as exemplary shown for the secretion machinery, are impaired in their functionality in absence of flotillins. These data provide first evidence that prokaryotic flotillins are elements that scaffold the plasma membrane and thereby provide a lipid environment that is vital for correct functionality of diverse cellular machineries.