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.