FRET-based Genetically Encoded Biosensor For Probing RsrA Function In Response To Redox Environment

The anti-oxidative activity in streptomyces coelicolor is induced by protein-protein interaction between a kind of RNA polymerase sigma factor σR and its corresponding zinc binding anti-sigma (ZAS) protein RsrA. RsrA bound to zinc associates with σR, inhibiting its transcriptional activity in a reducing environment. During disulfide stress RsrA forms intramolecular disulfide bonds, leading to zinc release and dissociation from aR, which initiates transcription to produce reductase and thioredoxin. Zinc in the RsrA-σR complex plays a crucial role in coordinating disulfide stress response mechanism, and the metal ligands tend to be Cys11, Cys41, Cys44and His37. Disulfide bond between Cys11and Cys44forms during disulfide stress, so that the zinc binding structure changes inducing the release of zinc ion.However, the oxidation process of RsrA is ambiguous. For further understanding the function of RsrA, we designed a fluorescence resonance energy transfer (FRET) based system, the RCP and SYP system, for real-time monitoring protein-protein interaction between σR and RsrA, and illustrating how this anti-oxidative stress system initiates in response to oxidants especially reactive oxygen species (ROS). According to signal changes, the FRET-based biosensor also confirms the function of zinc coordination and conformational change induced by ROS in RsrA. Sensitivity of RsrA-σR complex was determined in response to various ROS deriving from different metabolite pathways. The dissociation constant KD value obtained by FRET-based system was also verified by biolayer interferometry based analysis, and the conformational change of RCP was verified by far-UV circular dichroism. Based on the property of RsrA, another novel FRET-based single molecular biosensor YRCP was constructed and applied in ROS detection. For further application value, biosensor YRCP was co-expressed with an already constructed single fluorophore oxoglutarate detecting biosensor to create a novel multifunctional sensor bacterium.