Reengineering The Two-component Systems As Light-regulated In Escherichia Coli

Bacteria live in environments with dynamic changes. To sense and respond to different external stimuli, bacteria make use of numerous sensory-response circuits,called two-component systems (TCSs). A TCS comprises a histidine protein kinase(HK) sensing environmental stimuli and a response regulator protein (RR) regulating downstream genes. The two components are coupled via a phosphorylation control mechanism. As a new powerful tool, optogenetics perturbs and controls cellular and organismal behavior in a spatiotemporally exact manner by using genetically encoded,light-gated proteins, i.e. photoreceptors. Here, we adopt this approach to investigate TCSs by reengineering the sensor HKs as a light-sensing fusion protein. We constructed a light-controllable HK by replacing the signal-specific sensing domain of HK with the light-sensing domain of Cphl from Cyanobacteria Synechocystis, so that the HK can be investigated by red light. We constructed a series of HK-Cphl chimeras for the selected sixteen pairs of HK-RR TCSs in Escherichia coli. By taking the NarX-NarL system as an example, we demonstrated the light responsiveness of the constructed NarX-Cphl chimera and investigated the frequency response of the NarX-NarL system.