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. Author manuscript; available in PMC: 2023 Dec 1.
Published in final edited form as: Structure. 2022 Nov 14;30(12):1626–1636.e4. doi: 10.1016/j.str.2022.10.008

Figure 2. Characterization of the RhlR-PqsE interface and its role in RhlR-dependent signaling traits.

Figure 2.

a) Close-up view of the N-termini of RhlR (green) in the PqsE (purple) dimerization interface. Residues E189, R243, R246, R247 (purple) in the PqsE dimerization interface are highlighted. The N-termini (green) of RhlR are labeled. All structure images were created using Pymol. b) Close-up view of additional contacts between RhlR (green) and PqsE (purple). Residues R36, R37, and R154 on the RhlR side (green) of the RhlR-PqsE heterodimer interface are highlighted. Resides W142, E144, R170, R171, E206, R210, and E235 on the PqsE side (purple) of the RhlR-PqsE heterodimer interface are highlighted. All structure images were created using Pymol. c) Affinity purification pulldown of 6x-His-PqsE with E. coli lysates overexpressing WT RhlR and RhlR variants hypothesized to be involved in RhlR-PqsE complex formation. d) As in c) except the affinity purification pulldown was performed with WT 6x-His-PqsE and 6x-His-PqsE variants and E. coli lysates overexpressing WT RhlR. e) Pyocyanin production assay in a ΔrhlR strain containing empty pUCP18 plasmid or pUCP18 expressing WT RhlR or RhlR variants under the control of the rhlR promoter. All strains were normalized to the WT RhlR control set to 100% pyocyanin production. Three biological replicates were used, and each experiment was performed in technical duplicate. Error bars depict standard error of the mean for the biological replicates. P-values were determined by performing a one-way ANOVA with a Dunnett’s multiple comparisons test. f) qRT-PCR using the strains from e) to measure the transcript levels of hcnA and rhlA. All strains were normalized to WT RhlR using gyrA as an internal housekeeping control. Three biological replicates were used, and each experiment was performed in technical duplicate. Error bars depict standard error of the mean for the biological replicates. P-values were determined by performing multiple ratio paired t tests for each gene of interest within a given RhlR variant. See also Figure S4 and S5.