Investigation of protein interactions in the cell division complex of Bacillus subtilis reveals a possible mechanism for cell size homeostasis

Cell division is essential for bacterial survival. In this work, the cell division machinery, or divisome, of the gram positive, rod shaped bacterium Bacillus subtilis is investigated. The divisome is a protein complex consisting of many different subunits that sequentially and dynamically localize to the future division site. Four proteins assist the highly conserved tubulin homologue FtsZ to form a ring-like structure (Z-ring). After a delay, a second wave of proteins (late-proteins) are recruited to the division site, responsible for the synthesis of the crosswall between daughter cells. In this work, these dynamic interactions have been investigated using crosslinking mass-spectrometry (chapters 2 and 3) and fluorescence time-lapse microscopy (chapter 4). We developed a novel method allowing for direct crosslinking of cells in their growth medium. A proteome-wide analysis of crosslinked residues identified mainly abundant complexes, such as ribosomes and RNA polymerases. A more directed approach studied the interaction between FtsZ and the membrane anchor SepF in vitro. Based on the crosslinks identified and in silico docking, we propose that SepF dimers form a tetramer, which the FtsZ C-terminal peptide binds, possibly by being trapped between two SepF dimers. Finally, the delay between Z-ring formation and late-protein recruitment was addressed by time-lapse micrscopy, imaging fluorescent fusions of ZapA (Z-ring) and Pbp2B (late-protein). The highly variable delay negatively correlated with cell size and Z-ring concentration, and a size-dependent accumulation of Z-ring proteins was observed. This size-dependent Z-ring accumulation could be the molecular mechanism of cell size homeostasis in B. subtilis.