Figure 3.

Structural analysis of ZntR to guide design of the chimera MerRZntR^A29E. (A) Homology model of ZntR. I-TASSER⁵⁴ was used to generate a full-length homology model of ZntR. E. coli, accession code: AAC76317.1, C-score = 0.71, TM score = 0.91). The DNA-Binding Domain is indicated in mauve, while the C-terminal Domain comprising the metal binding loop is indicated in dark purple. Residues involved in interdomain communication (Glu-28, DNA-Binding Domain; Ser-43 and Arg-47, Metal-Binding Domain) are shown in lavender blue with hydrogen bonds shown in yellow. (B) Dose response behavior of the mutant MerRZntR^A29E following targeted mutagenesis of MerRZntR. Amino acid substitutions were introduced into the α-helix 2 and α-helix 3 loop region. The Environmental Protection Agency (EPA) guideline value is indicated, and the estimated limit of detection (LOD) for the regulatory circuit is shown (red). The most potent activator, MerRZntR^A29E is indicated in purple. (C) Metal-specificity of the MerRZntR^A29E based circuit. Inducers are colored using the key shown. For panels (B–C), cells were grown to OD₆₀₀ = ∼0.03 and induced with the concentrations of metals as indicated, with luciferase activity output (relative luminescence units [RLU]) normalized to optical density (OD₆₀₀) values (RLU/OD₆₀₀) for three time points (35, 40, and 45 min) postinduction. Values are presented as mean and ± standard deviation of either two or three independent replicates.