Characterizing Sequence−Function Relationships in Chimeric DcuS/EnvZ Histidine Kinases at Scale

Abstract

While bacterial sensor histidine kinases (SHKs) are widespread as natural molecular biosensors, tools for high-throughput characterization of SHK signaling phenotypes are limited, hindering widespread implementation of bacterial-based sensing. Here, we developed a synthetic two-component signaling system that reports chimeric SHK signaling through a standardized fluorescence readout. With this synthetic system, we screened a library of chimeric DcuS/EnvZ SHKs to characterize sequence-function relationships within the DcuS sensory and transmembrane domains. We quantified the effects of 1173 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 additionally reported a role for cytoplasmic N-terminal residues in determining SHK 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.