Sporulation-associated Mother Cell Lysis in Bacillus Displays Markers of Programmed Cell Death

Sporulation in Bacillus sp. requires the death of spore mother cell that has been hypothesized to be genetically programmed. However, there is lack of conclusive evidence supporting this hypothesis. The present study provides evidence showing expression of programmed cell death (PCD)-specific markers such as activation of caspase-3, externalization of phosphatidylserine (PS) detected by annexin V-FITC binding through flow cytometry, and damage to DNA evaluated by TUNEL assay during sporulation in B. subtilis and B. megaterium. Addition of cell-permeable irreversible inhibitor of caspase -3 was found to inhibit the sporulation process as also the caspase-3 activity and PS externalization. These findings were further revalidated using sporulation-deficient-mutants of B. subtilis, created using chemical mutagenesis. These mutants were found to be deficient in caspase-3 activity as well as the extent of PS externalization. Wildtype B. subtilis cells were found to have extracellular metal-dependent DNAse activity, which decreased in sporulation-deficient mutants. These finding provide evidence for the existence and association of markers of PCD during sporulation-associated mother cell lysis in Bacillus sp.