Combinatorial Regulation of clpC Expression During Sporulation in Bacillus subtilis

Abstract

Sporulation in the bacterium Bacillus subtilis is a tightly regulated developmental process triggered by stress or nutrient limitation. Sigma factors coordinate gene expression during this process by directing RNA polymerase to specific promoters. The clpC gene encodes an ATPase that partners with ClpP to degrade misfolded or unnecessary proteins, playing a crucial role in protein quality control and metabolic shutdown during sporulation. clpC is regulated by multiple sigma factor-dependent promoters and is expressed in the forespore, mother cell, and vegetative cells. Although essential for forming mature, heat-resistant spores, the contribution of each promoter to clpC expression and function remains unclear. To address this, I used CRISPR-Cas9 to generate deletion mutants targeting individual clpC promoters and a full upstream promoter knockout. A clpC-gfp reporter showed compartment-specific expression: one promoter drives forespore expression, another is active in the mother cell and vegetative cells, and a third has minimal activity. The full promoter knockout nearly eliminated clpC expression. Despite these differences, individual promoter deletions had no significant impact on sporulation efficiency, suggesting functional redundancy. The full promoter knockout generally sporulated normally but sometimes showed defects, indicating that very low clpC expression may suffice for sporulation or that compensatory mechanisms exist. Together, these findings highlight the complexity and flexibility of clpC regulation during sporulation.