Molecular Insight into the Regulation of Cell Differentiation by a Master Regulator Spo0A in starving Bacillus subtilis cells
Gram-positive soil bacterium Bacillus subtilis can activate cellular differentiation pathways such as biofilm formation and sporulation, in response to starvation. Spo0A, the master regulator of these cellular differentiation processes is activated (phosphorylated) via a multi-component phosphorelay signaling cascade comprising of histidine kinases (KinA, KinB, KinC) and phosphotransferases (Spo0F and Spo0B). A gradual increase in the Spo0A~P levels is crucial for proper initiation of these cellular differentiation processes and cell survival under starvation conditions. Despite decades of intensive research on these cellular processes, certain questions like how the cell fate decision to sporulate or form biofilms is made remain unanswered. However, when the cell decides to undergo the energy-consuming sporulation process, it forms a septum near one of the poles, resulting in two unequal compartments, larger mother cell, and a smaller forespore. After this asymmetric septation, Spo0A activity is found in the larger mother cell compartment, but its function and importance in sporulation remain unknown. Through this dissertation, I attempted to answer some of these fundamental questions connected to the master regulator Spo0A, such as the transcriptional control of spo0A and the mother cell-specific activity of Spo0A. Using EMSA, I provided the first of its kind direct evidence of Spo0A~P binding to three regulatory 0A boxes in the spo0A promoter region. Through transcriptional studies, I presented a revised model involving the role of 0A boxes in the spo0A transcription. I further found two previously undiscovered 0A boxes in the promoter region of spoIID, a mother cell-specific 0A-controlled gene. Transcriptional studies showed that spoIID expression is positively regulated by Spo0A~P binding to 0A1 and 0A2 boxes and repressed by Spo0A~P binding to 0A3 box. Through microscopy and immunoblot studies, I provided evidence that Spo0A~P regulation of spoIID expression is important for the completion of the engulfment process and SpoIID levels maintained by Spo0A~P regulation are crucial for proper sporulation. Based on these results, I propose a new model for spoIID expression governed by a triple-input AND gate consisting of σE-RNAP, SpoIIID, and Spo0A~P.