Structure and Composition of an Intercellular Channel in Sporulating Bacillus Subtilis

In response to nutrient starvation, the Gram-positive soil bacterium Bacillus subtilis forms a dormant spore in a process called sporulation. Sporulation involves an asymmetric cell division that results in a larger mother cell and a smaller forespore. The mother cell then ‘engulfs’ the forespore in a phagocytotic like mechanism and nurtures it before releasing it into the environment. Engulfment occurs in the sporulation pathway of the disease-causing spore forming bacteria Clostridium difficile and Bacillus anthracis and results in the reorganisation of the sporangium from two cells that lie by side to a cell within a cell structure. In this thesis, B. subtilis is used as a model organism to study engulfment to determine how bacterial cells localise proteins, move macromolecules and catalyse membrane fusion. Upon completion of engulfment, an intercellular channel is believed to form between the forespore protein SpoIIQ and the eight mother cell proteins encoded by the spoIIIAA-spoIIIAH operon. The channel is thought to allow the passage of a molecule important in forespore maturation, although the identity of this molecule is currently unknown. Whilst the structure of the complex formed between the extracellular C-terminal domains of SpoIIQ and SpoIIIAH has been determined by protein crystallography, no in-vivo experiments have been performed to address the stoichiometry of the channel complex in living cells. To determine the stoichiometry of the channel complex, high speed single molecule fluorescence microscopy was used to image live B. subtilis cells. Evidence was found to suggest that the channel complex assembles from SpoIIQ dimers and SpoIIIAH hexamers, suggesting that channel formation is more complicated than initially expected. SpoIIQ and SpoIIIAH were found to have low mobility and an average of 200-300 SpoIIQ and SpoIIIAH molecules were found in sporulating B. subtilis cells.