Bacillus subtilis Histidine Kinase KinC Activates Biofilm Formation by Controlling Heterogeneity of Single-Cell Responses

In Bacillus subtilis, biofilm and sporulation pathways are both controlled by a master regulator, SpoOA, which is activated by phosphorylation via a phosphorelay-a cascade of phosphotransfer reactions commencing with autophosphorylation of histidine kinases KinA, KinB, KinC, KinD, and KinE. However, it is unclear how the kinases, despite acting via the same regulator, SpoOA, differentially regulate downstream pathways, i.e., how KinA mainly activates sporulation genes and KinC mainly activates biofilm genes. In this work, we found that KinC also downregulates sporulation genes, suggesting that KinC has a negative effect on SpoOA activity. To explain this effect, with a mathematical model of the phosphorelay, we revealed that unlike KinA, which always activates SpoOA, KinC has distinct effects on SpoOA at different growth stages: during fast growth, KinC acts as a phosphate source and activates SpoOA, whereas during slow growth, KinC becomes a phosphate sink and contributes to decreasing SpoOA activity. However, under these conditions, KinC can still increase the population-mean biofilm matrix production activity. In a population, individual cells grow at different rates, and KinC would increase the SpoOA activity in the fast-growing cells but reduce the SpoOA activity in the slow-growing cells. This mechanism reduces single-cell heterogeneity of SpoOA activity, thereby increasing the fraction of cells that activate biofilm matrix production. Thus, KinC activates biofilm formation by controlling the fraction of cells activating biofilm gene expression. IMPORTANCE In many bacterial and eukaryotic systems, multiple cell fate decisions are activated by a single master regulator. Typically, the activities of the regulators are controlled posttranslationally in response to different environmental stimuli. The mechanisms underlying the ability of these regulators to control multiple outcomes are not understood in many systems. By investigating the regulation of Bacillus subtilis master regulator SpoOA, we show that sensor kinases can use a novel mechanism to control cell fate decisions. By acting as a phosphate source or sink, kinases can interact with one another and provide accurate regulation of the phosphorylation level. Moreover, this mechanism affects the cell-to-cell heterogeneity of the transcription factor activity and eventually determines the fraction of different cell types in the population. These results demonstrate the importance of intercellular heterogeneity for understanding the effects of genetic perturbations on cell fate decisions. Such effects can be applicable to a wide range of cellular systems.