Construction of a prototype two-component system from the phosphorelay system TodS/TodT.

Two-component systems (TCSs) play key roles in the adaptation of bacteria to environmental changes. In prototype TCSs a single phosphoryl transfer between the sensor kinase and response regulator occurs, whereas phosphorelay TCSs are characterised by a His1-Asp1-His2-Asp2 phosphorylation cascade. The TodS/TodT TCS controls the expression of a toluene degradation pathway and the TodS sensor kinase operates by a three-step internal phosphorelay. Based on TodS we report the construction of a minimal form of TodS, termed as Min-TodS, that contains only three of the seven TodS domains. Min-TodS is composed of the N-terminal PAS sensor domain as well as the C-terminal dimerisation/phosphotransfer domain and catalytic domain of TodS. We have conducted a comparative analysis of the phosphorelay TCS with its prototypal derivative. We demonstrate that Min-TodS binds effector molecules with affinities comparable with those observed for TodS. Min-TodS forms a TCS with TodT and toluene increases the amount of TodT-P. In contrast to TodS, toluene does not stimulate Min-TodS autophosphorylation. The half-life of Min-TodS-P was significantly increased as compared with TodS. Analysis of TodSD500A revealed that the hydrolysis of the acylphosphate of the receiver domain is responsible for the reduced half-life of TodS. The regulation of P(todX) expression by Min-TodS/TodT and TodS/TodT in response to different effectors are compared. The Min-TodS/TodT system was characterized by a higher basal activity but a lower magnitude of response. Data will be discussed in the context that the phosphorelay system appears to be better suited for the control of a degradation pathway for toxic compounds.