Sporulation in Bacillus subtilis is controlled by a complex gene regulatory circuit that is activated upon nutrient deprivation. The initial process is directed by the phosphorelay, involving the major sporulation histidine kinase (KinA) and two additional phosphotransferases (Spo0F and Spo0B), that activates the master transcription factor Spo0A. Little is known about the initial event and mechanisms that trigger sporulation. Using a strain in which the synthesis of KinA is under the control of an IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible promoter, here we demonstrate that inducing the synthesis of the KinA beyond a certain level leads to the entry of the irreversible process of sporulation irrespective of nutrient availability. Moreover, the engineered cells expressing KinA under a H -dependent promoter that is similar to but stronger than the endogenous kinA promoter induce sporulation during growth. These cells, which we designated COS (constitutive sporulation) cells, exhibit the morphology and properties of sporulating cells and express sporulation marker genes under nutrient-rich conditions. Thus, we created an engineered strain displaying two cell cycles (growth and sporulation) integrated into one cycle irrespective of culture conditions, while in the wild type, the appropriate cell fate decision is made depending on nutrient availability. These results suggest that the threshold level of the major sporulation kinase acts as a molecular switch to determine cell fate and may rule out the possibility that the activity of KinA is regulated in response to the unknown signal(s).

Starving Bacillus subtilis cells execute a gene expression program resulting in the formation of stress-resistant spores. Sporulation master regulator, Spo0A, is activated by a phosphorelay and controls the expression of a multitude of genes, including the forespore-specific sigma factor σ F and the mother cell-specific sigma factor σ E . Identification of the system-level mechanism of the sporulation decision is hindered by a lack of direct control over Spo0A activity. This limitation can be overcome by using a synthetic system in which Spo0A activation is controlled by inducing expression of phosphorelay kinase KinA. This induction results in a switch-like increase in the number of sporulating cells at a threshold of KinA. Using a combination of mathematical modeling and single-cell microscopy, we investigate the origin and physiological significance of this ultrasensitive threshold. The results indicate that the phosphorelay is unable to achieve a sufficiently fast and ultrasensitive response via its positive feedback architecture, suggesting that the sporulation decision is made downstream. In contrast, activation of σ F in the forespore and of σ E in the mother cell compartments occurs via a cascade of coherent feed-forward loops, and thereby can produce fast and ultrasensitive responses as a result of KinA induction. Unlike σ F activation, σ E activation in the mother cell compartment only occurs above the KinA threshold, resulting in completion of sporulation. Thus, ultrasensitive σ E activation explains the KinA threshold for sporulation induction. We therefore infer that under uncertain conditions, cells initiate sporulation but postpone making the sporulation decision to average stochastic fluctuations and to achieve a robust population response.cell fate | development | differentiation | stochasticity | network I n response to nutrient deprivation, Bacillus subtilis cells undergo asymmetrical cell division and then follow a cell differentiation program resulting in formation of metabolically inert spores (1, 2) (Fig. 1A). Sporulation requires the execution of a complex gene expression program involving hundreds of "sporulation" genes (3-6). The availability of a large number of genetic mutants that differ from the WT only in their sporulation response makes B. subtilis an ideal model system to study the relationship between gene expression and cell fate specification during bacterial differentiation (7).Progression of the sporulation program is under the control of a large regulatory network (hereafter called the sporulation network). This network involves the sporulation master regulator Spo0A and five alternative sigma factors (σ H , σ F , σ E , σ K , and σ G ) that are activated in precise temporal order (8). Initiation of the sporulation program is controlled by Spo0A (4, 9). The activity and concentration of this master transcription factor are regulated by phosphorelay through both posttranslational and transcriptional interactions (10). Posttranslationally, phosphoryl groups are transferred fro...

SummarySporulation initiation in Bacillus subtilis is controlled by the phosphorylated form of the master regulator Spo0A which controls transcription of a multitude of sporulation genes. In this study, we investigated the importance of temporal dynamics of phosphorylated Spo0A (Spo0A∼P) accumulation by rewiring the network controlling its phosphorylation. We showed that simultaneous induction of KinC, a kinase that can directly phosphorylate Spo0A, and Spo0A itself from separately controlled inducible promoters can efficiently trigger sporulation even under nutrient rich conditions. However, the sporulation efficiency in this artificial two-component system was significantly impaired when KinC and/or Spo0A induction was too high. Using mathematical modelling, we showed that gradual accumulation of Spo0A∼P is essential for the proper temporal order of the Spo0A regulon expression, and that reduction in sporulation efficiency results from the reversal of that order. These insights led us to identify premature repression of DivIVA as one possible explanation for the adverse effects of accelerated accumulation of Spo0A∼P on sporulation. Moreover, we found that positive feedback resulting from autoregulation of the native spo0A promoter leads to robust control of Spo0A∼P accumulation kinetics. Thus we propose that a major function of the conserved architecture of the sporulation network is controlling Spo0A activation dynamics.

In response to starvation, Bacillus subtilis cells differentiate into different subsets, undergoing cannibalism, biofilm formation or sporulation. These processes require a multiple component phosphorelay, wherein the master regulator Spo0A is activated upon phosphorylation by one or a combination of five histidine kinases (KinA-KinE) via two intermediate phosphotransferases, Spo0F and Spo0B. In this study, we focused on KinC, which was originally identified as a sporulation kinase and was later shown to regulate cannibalism and biofilm formation. First, genetic experiments using both the domesticated and undomesticated (biofilm forming) strains revealed that KinC activity and the membrane localization are independent of both the lipid raft marker proteins FloTA and cytoplasmic potassium concentration, which were previously shown to be required for the kinase activity. Next, we demonstrated that KinC controls cannibalism and biofilm formation in a manner dependent on phosphorelay. For further detailed characterization of KinC, we established an IPTG-inducible expression system in the domesticated strain, in which biofilm formation is defective, for simplicity of study. Using this system, we found that the N-terminal transmembrane domain is dispensable but the PAS domain is needed for the kinase activity. An in vivo chemical cross-linking experiment demonstrated that the soluble and functional KinC (KinC DTM1+2 ) forms a tetramer. Based on these results, we propose a revised model in which KinC becomes active by forming a homotetramer via the N-terminal PAS domain, but its activity is independent of both the lipid raft and the potassium leakage, which was previously suggested to be induced by surfactin.

Upon starvation, Bacillus subtilis cells switch from growth to sporulation. It is believed that the N-terminal sensor domain of the cytoplasmic histidine kinase KinA is responsible for detection of the sporulation-specific signal(s) that appears to be produced only under starvation conditions. Following the sensing of the signal, KinA triggers autophosphorylation of the catalytic histidine residue in the C-terminal domain to transmit the phosphate moiety, via phosphorelay, to the master regulator for sporulation, Spo0A. However, there is no direct evidence to support the function of the sensor domain, because the specific signal(s) has never been found. To investigate the role of the N-terminal sensor domain, we replaced the endogenous three-PAS repeat in the N-terminal domain of KinA with a two-PAS repeat derived from Escherichia coli and examined the function of the resulting chimeric protein. Despite the introduction of a foreign domain, we found that the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. Further, by using chemical cross-linking, we showed that the chimeric protein exists predominantly as a tetramer, mediated by the N-terminal domain, as was found for KinA. These results suggest that tetramer formation mediated by the N-terminal domain, regardless of the origin of the protein, is important and sufficient for the kinase activity catalyzed by the C-terminal domain. Taken together with our previous observations, we propose that the primary role of the N-terminal domain of KinA is to form a functional tetramer, but not for sensing an unknown signal.Bacterial cells are directly exposed to the environment. Hence, they must sense and respond rapidly to changes in their local environment in order to survive. One prevailing strategy to overcome this problem is to utilize the two-component system composed of a sensor histidine kinase (HK) and a response regulator (RR) (35,41). In the most typical case, the HK is a transmembrane protein with the N-terminal sensor domain often situated in the extracytoplasmic compartment, such as the periplasm, inner or outer membrane, or even extracellular space, and the C-terminal autokinase domain, containing a conserved phospho-accepting histidine residue, normally resides in the cytosol (8,35,41). In contrast, the RR comprises a regulatory domain that includes a phospho-accepting aspartate residue at the N terminus, followed by an associated effector domain typically containing a DNA-binding motif, so that it becomes active as a transcription factor upon phosphorylation. Thus, using these two components, various environmental signals can be detected by the HK and transmitted as phosphate groups to the RR for cellular adaptation.The individual sensor domains of HKs are highly variable and thus lack sequence homology with other HKs (8,44,45). Therefore, it is believed that a variety of environmental signals can be detected, with a high degree of sensiti...