The master regulator for entry into sporulation in Bacillus subtilis becomes a mother cell-specific transcription factor for forespore engulfment.

The Spo0A transcription factor is activated by phosphorylation in starving Bacillus subtilis cells. The activated Spo0A (Spo0A~P) regulates genes controlling entry into sporulation and appears to control mother-cell-specific gene expression after asymmetric division, but the latter remains elusive. Here, we found that Spo0A~P directly binds to three conserved DNA sequences (0A1-3) in the promoter region of the mother cell-specific lytic transglycosylase gene spoIID, which is transcribed by σE -RNA polymerase (RNAP) and negatively controlled by the SpoIIID transcription factor and required for forespore engulfment. Systematic mutagenesis of the 0A boxes revealed that the 0A1 and 0A2 boxes located upstream of the promoter positively control the transcription of spoIID. In contrast, the 0A3 box located downstream of the promoter negatively controls the transcription of spoIID. The mutated SpoIIID binding site located between the -35 and -10 promoter elements causes increased expression of spoIID and reduced sporulation. When the mutations of 0A1, 0A2, and IIID sites are combined, sporulation is restored. Collectively, our data suggest that the mother cell-specific spoIID expression is precisely controlled by the coordination of three factors, Spo0A~P, SpoIIID, and σE -RNAP, for proper sporulation. The conservation of this mechanism across spore-forming species was discussed.

The Slowdown of Growth Rate Controls the Single-Cell Distribution of Biofilm Matrix Production via an SinI-SinR-SlrR Network.

In Bacillus subtilis, master regulator Spo0A controls several cell-differentiation pathways. Under moderate starvation, phosphorylated Spo0A (Spo0A~P) induces biofilm formation by indirectly activating genes controlling matrix production in a subpopulation of cells via an SinI-SinR-SlrR network. Under severe starvation, Spo0A~P induces sporulation by directly and indirectly regulating sporulation gene expression. However, what determines the heterogeneity of individual cell fates is not fully understood. In particular, it is still unclear why, despite being controlled by a single master regulator, biofilm matrix production and sporulation seem mutually exclusive on a single-cell level. In this work, with mathematical modeling, we showed that the fluctuations in the growth rate and the intrinsic noise amplified by the bistability in the SinI-SinR-SlrR network could explain the single-cell distribution of matrix production. Moreover, we predicted an incoherent feed-forward loop; the decrease in the cellular growth rate first activates matrix production by increasing in Spo0A phosphorylation level but then represses it via changing the relative concentrations of SinR and SlrR. Experimental data provide evidence to support model predictions. In particular, we demonstrate how the degree to which matrix production and sporulation appear mutually exclusive is affected by genetic perturbations. IMPORTANCE The mechanisms of cell-fate decisions are fundamental to our understanding of multicellular organisms and bacterial communities. However, even for the best-studied model systems we still lack a complete picture of how phenotypic heterogeneity of genetically identical cells is controlled. Here, using B. subtilis as a model system, we employ a combination of mathematical modeling and experiments to explain the population-level dynamics and single-cell level heterogeneity of matrix gene expression. The results demonstrate how the two cell fates, biofilm matrix production and sporulation, can appear mutually exclusive without explicitly inhibiting one another. Such a mechanism could be used in a wide range of other biological systems.

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, Spo0A, 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, Spo0A, 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 Spo0A activity. To explain this effect, with a mathematical model of the phosphorelay, we revealed that unlike KinA, which always activates Spo0A, KinC has distinct effects on Spo0A at different growth stages: during fast growth, KinC acts as a phosphate source and activates Spo0A, whereas during slow growth, KinC becomes a phosphate sink and contributes to decreasing Spo0A 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 Spo0A activity in the fast-growing cells but reduce the Spo0A activity in the slow-growing cells. This mechanism reduces single-cell heterogeneity of Spo0A 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 Spo0A, 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.