Study of Histidine Kinases Involved in Bacillus substilis Differentiation

In response to nutrient deprivation, Bacillus subtilis cells undergo differentiation to form biofilm and spore. This process requires the involvement of a multiple phosphorelay component, wherein phosphate is transferred to a master transcription regulator Spo0A from five histidine kinases (KinA-KinE) via two intermediate phosphotransferases, Spo0F and Spo0B. It has been proposed that the autokinase activity of one or a combination of five kinases is stimulated when the amino (N) terminal ‘sensor’ domain of kinase receives an yet unidentified starvation signal(s), leading to the increase of the phosphorylated (active) form of Spo0A (Spo0A~P) via phosphorelay and resulting in differentiation into biofilm formation and sporulation. However, the underlying mechanisms by which the putative starvation signal(s) stimulate the kinase and the subsequent control of Spo0A activity remain elusive. In order to test, we conducted, domain-swap experiments were performed by fusing a tetramer-forming protein derived from Escherichia coli to the KinA C-terminal domain. Despite the introduction of a foreign domain, the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. A simultaneous induction system, in which KinC, a kinase that can directly phosphorylate Spo0A, and Spo0A itself are separately controlled by inducible promoters, were constructed. This artificial two-component system can efficiently trigger sporulation even under nutrient rich conditions. However, the sporulation efficiency was significantly impaired when KinC and/or Spo0A induction was too high. Lastly, the role of KinC during the course of starvation was studied. Conventional genetic approaches showed that KinC preferentially and positively controls the expression of the cannibalism (a mechanism to delay sporulation) genes during early stage of starvation in a manner dependent on phosphorelay, resulting in the delay of sporulation. Evidence suggests that the N-terminal domain is essential for forming a stable tetramer as a functional kinase, but possibly not for sensing an as-yet unknown starvation signal. Furthermore, the data suggest that, upon starvation, two different levels (low and high) of Spo0A activity are achieved through two distinct pathways regulated by KinC and KinA, which are essential for the proper cell fate decision to differentiate into biofilm or sporulation.