Characterizing Sequence-Function Relationships in Chimeric DcuS/EnvZ Histidine Kinases at Scale
While bacterial sensor histidine kinases (SHKs) are widespread as natural molecular biosensors, tools for high-throughput characterization of SHK signaling phenotypes are limited, hindering wide scale implementation of bacterial-based sensing. Here, we developed a synthetic two-component signaling system that reports chimeric SHK signaling via a standardized fluorescence readout. With this synthetic system, we screened a library of chimeric DcuS/EnvZ SHKs to characterize sequence-function relationships within in the DcuS sensory and transmembrane domains. We quantified the effects of 1,173 mutations on signaling outputs in the presence of fumarate, a native DcuS ligand, as well as aspartate for which DcuS has minimal affinity for. We identified eleven positions across the DcuS domains which significantly alter aspartate responsiveness and selectivity and further observed a role for cytoplasmic N-terminal residues in determining signaling outputs. In future studies, this framework will expedite design of biosensors for novel ligands by enabling high-throughput screening of mutagenized libraries of natural SHKs.