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. 2022 Dec 1;13:7402. doi: 10.1038/s41467-022-35030-w

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Western blot demonstrating the synergistic solubility-enhancing effect of C4-HSL and PqsE on RhlR. RhlR and His₆-PqsE were expressed from plasmid vectors in E. coli BL21-CodonPlus(DE3)-RIL in the presence of the indicated concentrations of C4-HSL. Soluble extracts were analyzed using GroEL as a control. The marker (M) indicates the molecular weight in kDa. A typical result from at least three independent experiments is shown. The corresponding whole-cell extracts are shown in Supplementary Fig. 1a. b SDS-PAGE of a co-expression analysis of His₆-tagged PqsE and RhlR in E. coli BL21-CodonPlus(DE3)-RIL. Induced cells were harvested, lysed and separated into an insoluble (P) and a soluble fraction (S), which was used to incubate Ni-NTA beads, washed and then boiled in SDS-loading buffer (B). The marker (M) indicates the molecular weight in kDa. A typical result from at least three independent experiments is shown. c Time-dependent stability analysis of PqsE/RhlR:C4-HSL and RhlR:mBTL. Purified proteins were incubated at 37 °C and the soluble fraction was analyzed by SDS-PAGE after the indicated period. The marker (M) indicates the molecular weight in kDa. A typical result from at least three independent experiments is shown. d SEC-MALS analysis of RhlR purified in the presence of mBTL, of PqsE and of the PqsE/RhlR:C4-HSL complex. The molecular mass of PqsE suggests that the protein is dimeric. PqsE and RhlR interact in a 2:2 complex. e Proteomic analysis of a pulldown with His₆-tagged PqsE in P. aeruginosa PA14 vs. a His₆-FlgZ control. Buffers were supplemented with 50 µM C4-HSL. Proteins with more than 50% sequence coverage in the analysis are shown. Log₂-fold enrichments larger than 3 (Ni-NTA bead eluate vs. whole-cell extract) are colored in red and green, respectively. The analysis confirms the interaction of PqsE and RhlR in P. aeruginosa. Source data are provided as a Source Data file.