Figure 3. Contextualizing the RhlR-PqsE interaction in QS.

a) left) Overlay of the structures of SdiA (pink; PDB: 4LFU³⁹) and RhlR (green); right) comparison of the DBD of TraR (brown; PDB: 1H0M⁴⁰) and RhlR (green) bound to their respective tra or rhl boxes. b) E. coli reporter assay expressing LasR under the control of the pBAD promoter in the presence of the lasB promoter fused to luxCDABE with (purple) or without (orange) constitutively expressed PqsE. The assay was performed in a 10-point dose response with 2-fold serial dilutions. The top concentration of 3OC₁₂HSL was 2 nM. Three biological replicates were used, and each experiment was performed in technical duplicate. Error bars depict standard error of the mean. RLU = relative light units. A non-linear regression analysis was performed for each of the curves and a P value was assigned based on differences in the calculated EC₅₀ value. c) Ribbon diagram depicting the conservation analysis of RhlR compared to 150 LuxR-type receptors. One monomer is colored in rainbow based on conservation scores from ConSurf analysis shown in Table S1 and the other monomer is colored gray for clarity. Red residues are highly conserved; blue residues are highly variable amongst the 150 homologs analyzed. PqsE is omitted for clarity. d) Overlay of the structure of LasR (orange; PDB: 6MWL⁴⁵) with RhlR (green) at the PqsE (purple) interface. Residues D23 and T144 in LasR (orange) and residue R37 and R154 in RhlR (green) are highlighted. LasR and RhlR are depicted as ribbons for clarity of the overlaid structures. All structure images were created using Pymol. See also Figure S6 and Table S1.