Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans.

UNLABELLED: Entry into sporulation in Bacillus subtilis is governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur in trans. To test this hypothesis, we generated a series of B. subtilis strains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in a trans fashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressed in vivo. Taken together, these in vitro and in vivo results reinforce the evidence that KinA autophosphorylation is able to occur in a trans fashion. IMPORTANCE: Autophosphorylation of histidine kinases is known to occur by either the cis (one subunit of kinase phosphorylating itself within the multimer) or the trans (one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided direct in vivo and in vitro evidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur in trans within the homotetramer complex. While the physiological and mechanistic significance of the trans autophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion.

In vivo functional characterization of the transmembrane histidine kinase KinC in Bacillus subtilis.

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(ΔTM1+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.

Triggering sporulation in Bacillus subtilis with artificial two-component systems reveals the importance of proper Spo0A activation dynamics.

Sporulation 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.

Expression level of a chimeric kinase governs entry into sporulation in Bacillus subtilis.

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.

The threshold level of the sensor histidine kinase KinA governs entry into sporulation in Bacillus subtilis.

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-beta-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 sigma(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).