The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for the autokinase activity

Sporulation in Bacillus subtilis is induced upon starvation. In a widely accepted model, an N‐terminal “sensor” domain of the major sporulation kinase KinA recognizes a hypothetical starvation signal(s) and autophosphorylates a histidine residue to activate the master regulator Spo0A via a multicomponent phosphorelay. However, to date no confirmed signal has been found. Here, we demonstrated that PAS‐A, the most N‐terminal of the three PAS domains (PAS‐ABC), is dispensable for the activity, contrary to a previous report. Our data indicated that the autokinase activity is dependent on the formation of a functional tetramer, which is mediated by, at least, PAS‐B and PAS‐C. Additionally, we ruled out the previously proposed notion that NAD (+)/NADH ratio controls KinA activity through the PAS‐A domain by demonstrating that the cofactors show no effects on the kinase activity in vitro. In support of these data, we found that the cofactors exist in approximately 1000‐fold excess of KinA in the cell and the cofactors’ ratio does not change significantly during growth and sporulation, suggesting that changes in the cofactor ratio might not play a role in controlling KinA activity. These data may refute the widely‐held belief that the activity of KinA is regulated in response to an unknown starvation signal(s).