Fig. 4.

Engineering NarX chimeras for expanded sensing capabilities. (A) Membrane-bound histidine kinases generally possess similar structural features, allowing chimeric proteins with new functions to be engineered by mixing and matching parts. (B) Sequence alignment of NarX (nitrate), NrsS (nickel), RssA (iron), and VanS (vancomycin) HAMP domains. Asterisk (*), colon (:), and periods (.) indicate fully conserved amino acids, or amino acids with strongly conserved, and weakly conserved properties, respectively. The red arrow indicates the cross-over point of chimeras. (C–E) Luminescence as a result of NrsS, RssA, and VanS chimera signaling when expressed in POPC, DOPC, or DMPC membranes with each chimera’s respective ligand. Fold change in luminescence in response to each ligand relative to water is indicated above each membrane composition. (F) NarX and NrsS activate specifically to their respective ligands. (G and H) Coexpressing NarX and the NrsS chimera allows for sensing of nitrate and nickel in POPC vesicles. All error bars represent the SEM for n = 3 independent replicates.