Molecular dynamics guided analysis of Bacillus subtilis spore germination mechanisms

Some species within the Bacillales and Clostridiales orders form spores under unfavourable environmental conditions. These spores are metabolically dormant and highly resistant to extreme stress. The spore core, analogous to the protoplast of vegetative cells, contains only 25–45% water by wet weight, compared to approximately 80% in vegetative cells. Upon activation by small-molecule nutrients, spores germinate, restoring core water content and metabolism while becoming susceptible to killing, before progressing through outgrowth to vegetative growth. GerAB is the B subunit of the prototypical Bacillus subtilis GerA germinant receptor (GR), a membrane protein belonging to the Amino Acid–Polyamine–Organocation (APC) superfamily of transporters. It functions as the L-alanine sensor that initiates germination. Based on molecular dynamics (MD) simulations, GerAB was previously predicted to contain a putative water channel. Using a combination of MD simulations, steered MD (SMD) simulations, and mutagenesis, this study investigates the function of GerAB in high molecular detail. To be specific, we first investigated putative water channel function of GerAB, identified key residues through MD and SMD simulations, and verified their functional roles in vivo with mutagenesis. We further elucidated a potential mechanism of GerAB water channel opening using a combination of MD and SMD. Together, these findings extend the current understanding of GR function through an integrated in silico and in vivo approach. The interdisciplinary methodology developed in this thesis was also incorporated into an undergraduate education module.