Investigation of clpP Expression During Bacillus subtilis Sporulation

Abstract

Sporulation in Bacillus subtilis is highly regulated by stage-specific sigma factors that control gene expression as the cell transitions to a metabolically dormant state. The AAA+ protease complex ClpCP, activated in the forespore by the adaptor protein MdfA, plays an important role in degrading metabolites during metabolic shutdown. Recent findings in our lab and others demonstrate that clpC is turned on by the forespore-specific sigma factor, sigF, as is mdfA, raising the possibility that clpP is similarly regulated. Additionally, bioinformatic analysis of the region upstream of clpP reveal matching to consensus sequences for both sigF and the stress-activated sigB, in line with other dual-activated promoters in B. subtilis. We also identified a match to a sigE consensus sequence nearby. My project aimed to test the hypothesis that clpP expression during sporulation is controlled by a sigF-activated promoter. In order to evaluate this, we constructed a PclpP-lacZ fusion and transformed it into IPTG-inducible sigF, sigB or sigE strains and measured beta-galactosidase activity. We found that the PclpP promoter is activated by sigF, but not by sigE. This project helped advance our understanding of sporulation in B. subtilis and provided insight into conserved regulatory mechanisms in pathogenic bacteria.