Systematic domain deletion analysis of the major sporulation kinase in Bacillus subtilis.

When Bacillus subtilis cells encounter nutrient deprivation, they decide to switch from a symmetric cell division to an asymmetric cell division as a means of survival. The complex signal transduction system includes at least three sensor histidine kinases (KinA, KinB, and KinC), two phosphotransferases (Spo0F and Spo0B), and one response regulator (Spo0A). Although KinD and KinE were originally suggested to be sensor kinases involved in sporulation (11), an in vivo study demonstrated that these kinases do not feed phosphates into the phosphorelay under physiological conditions (6). Among the three sensor kinases, the major sporulation kinase is the cytoplasmic protein KinA (17). Upon nutrient limitation, KinA autophosphorylates and indirectly transfers the phosphate group to the master transcription factor Spo0A via two intermediate proteins, Spo0F and Spo0B (1). Once the activated Spo0A accumulates to a certain threshold level in a gradual manner, each of the 121 genes under the direct control of the master regulator is turned on or off at different times depending on the concentration of the regulator (5, 15). Therefore, the four-component phosphate transfer system found in B. subtilis sporulation is called a multicomponent “phosphorelay” (1, 9). Based on the structural and functional characterization of other two-component systems, mostly in Escherichia coli, it has been defined that amino-terminal (N-terminal) and carboxy-terminal (C-terminal) domains are sensor and autokinase domains, respectively (18, 20, 25, 26). In KinA, the N-terminal sensor domain contains three PAS domains (PAS-A, -B, and -C) (14, 20, 25), and the C-terminal autokinase domain includes a dimerization and histidine phosphotransfer (DHp) domain and a catalytic ATP-binding (CA) domain (18, 25). The PAS domain was originally found in the Drosophila period clock protein (PER), the vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and the Drosophila single-minded protein (SIM) as imperfect repeat sequences involved in protein-protein interaction (2, 16). PAS domains have been found in numerous histidine kinases of two-component systems in bacteria and are suggested to be responsible for sensing a variety of signals and regulate the activity of each of the associated histidine kinases (8, 22, 23). Thus, it has been widely believed that PAS domains of KinA could sense an as-yet-unknown sporulation signal and activate the sporulation kinase through autophosphorylation (14, 20, 25). However, the signaling molecule that is sensed by the PAS domains in KinA has never been identified. Therefore, the molecular mechanism by which KinA gets activated is still not clear. In our recent report, using our artificial sporulation induction (ASI) system (see Fig. S1 in the supplemental material), which triggers sporulation in response to the artificial induction of KinA (6), it has been shown that the PAS-A domain of KinA, which was originally believed to be essential for signal sensing and function (14, 20, 25), is dispensable (3). It has also been suggested that the role of PAS domains in KinA is to form a stable homotetramer complex and possibly not for sensing the starvation-induced signal (3). Here in this study, we furthered our understanding of the role of PAS domains by constructing a series of systematic PAS domain deletion mutants consisting of a different combination of domain deletions and studying them individually.