Decoding the Sporulation Process in Bacillus Subtilis

Bacillus subtilis undergoes the process of sporulation under nutrient poor conditions. The initiation of sporulation begins with the autophosphorylation of KinA, the major sporulation kinase. While it is widely accepted that an as-yet-unknown starvation signal(s) triggers the autophosphorylation through interaction with the N-terminal PAS-domain of KinA, there has been no definitive identification of such a signal. Here, I suggest that the autokinase activity of KinA is dependent on tetrameric formation of the kinase which occurs upon reaching a certain threshold. The following autophosphorylation, a phosphorelay occurs in a complex version of a two-component response regulator system ending with the phosphorylation of Spo0A, the master regulator of sporulation. A certain threshold of Spo0A~P must be reached to enter into sporulation and Spo0A~P levels have been found to increase in the mother cell compartment even after septation. I investigated the control of Spo0A~P on sporulation specific genes by use of a Spo0A inhibitor. I found that expression of a competitive Spo0A inhibitor within the mother-cell compartment resulted in a decrease in activity of specific sporulation reporter genes. This effect was greatly seen at the late stage reporter (PgerE). RNA-Seq analysis of the late stage reporter under sporulation conditions reported several significant Spo0A-controlled genes during sporulation. Notably, genes (ldh and lctP) involved in overflow metabolism and fermentation were significantly upregulated. Finally, I performed preliminary studies on three known Spo0A-controlled genes spoIID, spoIIE, and divIVA. The levels of SpoIID within the cell were found to be moderated by a triple AND gate controlling the expression of spoIID. SpoIIE and DivIVA protein expression was observed by fluorescent microscopy in the presence of the Spo0A inhibitor to determine the effect of the inhibitor on protein localization. My initial results showed abnormal localization patterns for each. These studies may lead to a greater understanding of the regulations of control by Spo0A during sporulation.