The Master Regulator Spo0A Integrates DNA Sequence and Shape to Regulate the Sporulation Kinase Gene kinA in Bacillus...

Sporulation in starved Bacillus subtilis depends on precise control of kinA, which encodes the major phosphorelay kinase that generates the threshold level of phosphorylated Spo0A (Spo0A~P) required for entry into sporulation. Here, we define the regulatory logic that shapes kinA expression and uncover structural features that influence Spo0A~P recruitment. First, we show that the kinA promoter integrates positive and negative Spo0A~P inputs through a composite three-site 0A-box architecture. Distinct affinities and cooperative interactions among the three sites generate a controlled output in which activating and repressive inputs are balanced during starvation, maintaining basal-to-moderate kinA transcription. Second, we identify intrinsic DNA bending as a sequence-independent mechanism that enhances Spo0A~P engagement, promoting low-specificity interactions that may contribute to transcriptional regulation and more broadly chromosome organization. Finally, we show that both the kinA promoter and KinA protein domains diverge between pathogenic and nonpathogenic Bacillus species, reflecting niche-specific evolutionary pressures on sporulation initiation. Together, these findings support an integrated model in which promoter architecture and DNA shape collectively tune Spo0A~P dependent regulation of kinA.