Fig 1.
Cartoon schematic of quorum sensing in Vibrio cholerae.
Quorum sensing in V. cholerae is regulated by four receptor histidine kinases: CqsR, LuxPQ, CqsS, and VpsS. Top: Low-cell density signaling. All four QS receptors act as histidine kinases, phosphorylating LuxU, which transfers a phosphoryl group to LuxO and leads to transcription of Qrr1-4. These small RNAs inhibit HapR and induce AphA production, respectively. Bottom: High-cell density signaling. The QS receptors bind their respective autoinducers, inhibiting receptor kinase activity. This causes phosphoryl group transfer away from LuxO, resulting in decreased Qrr1-4 transcription and promoting HapR production. Created in BioRender. Guarnaccia, A. (2025) https://BioRender.com/tfg4c8z.
Table 1.
Data collection and refinement statistics.
Fig 2.
CqsRp-ethanolamine crystal structure.
(A) Left: The CqsRp monomer and ethanolamine are rendered as a cyan cartoon and spheres, respectively. The schematic was produced using PyMOL (PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC). Right: Expanded view of the area enclosed by the box in the left panel. Fo-Fc electron density corresponding to ethanolamine scaled to 3σ. Positive electron density is shown in green and negative density shown in red. Fo-Fc electron density was calculated prior to ligand and water building. The schematic was produced using Coot [34]. (B) Ethanolamine (ball-and-stick model) and its coordinating residues (cyan sticks) are shown. The schematic was produced using PyMOL (PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC). (C) Schematic representation of CqsRp-ethanolamine binding. CqsRp and ethanolamine are depicted as blue and grey bonds, respectively. Hydrogen bonds are depicted as black dashed lines. Hydrophobic contacts are depicted as lines radiating from the semicircles and spheres. The schematic was produced using LigPlot+ [35].
Fig 3.
MST quantification of CqsRp binding to ethanolamine, L-alaninol, and serinol.
(A) Ethanolamine was titrated between 500.0 μM and 15.3 nM with 100.0 nM CqsRp. MST was performed in triplicate. (B) L-alaninol was titrated between 1.0 mM and 30.5 nM with 100.0 nM CqsRp. MST was performed in quadruplicate. (C) Serinol was titrated between 5.0 mM and 152.6 nM with 100.0 nM CqsRp. MST was performed in quadruplicate.
Fig 4.
Dissociation constants derived from MST studies of CqsRp binding to ethanolamine analogs.
Reported dissociation constants (Kd) for ethanolamine and compounds of similar chemical composition. Diagrams of each respective structure are shown. Nb indicates that no binding was detected.
Fig 5.
CqsRp-L-alaninol and CqsRp-serinol binding sites.
(A and B) CqsRp-L-alaninol and CqsRp-serinol binding-site residues are depicted as balls and sticks. Fo-Fc maps scaled to 3σ with positive density shown in green and negative density shown in red. Fo-Fc electron density was calculated prior to ligand and water building. The schematic was produced using Coot [34]. (C and D) Isolated views of the respective ligand binding sites. CqsR residues that coordinate L-alaninol or serinol are shown as orange or green sticks, respectively. Ligands are shown as ball-and-stick models. Note the acidic platform residue Asp198, the hydrophilic residues below the ligands, and the hydrophobic residues forming the lid above. The schematic was produced using PyMOL (PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC). (E and F) Schematic representation of CqsRp-L-alaninol and CqsRp-serinol binding. CqsRp and L-alaninol are depicted as orange and gray bonds, respectively, in panel E. CqsRp and serinol are depicted as green and gray bonds, respectively, in panel F. Hydrogen bonds are depicted as black dashed lines. Hydrophobic contacts are depicted as lines radiating from the semicircles and spheres. The schematic was produced using LigPlot+ [35].
Fig 6.
Response to exogenously added ethanolamine by different CqsR variants in V. cholerae.
Whole-cell bioluminescence assays were performed using a ∆cqsS ∆luxPQ ∆cqsR ∆vpsS strain with different CqsR variants expressed from a plasmid. These strains also contain a Pqrr4-luxCDABE reporter to measure the transcription of qrr4. Ethanolamine was added at the final concentrations as shown. Curves of the same color represent bacterial cultures grown with the same concentration of ethanolamine. For comparison, the two dotted lines on each graph show the maximum light production level of WT in the absence of ethanolamine and the minimal light production level of WT in the presence of the highest amount of ethanolamine at low cell density (OD600 ~ 0.2). Representative results are shown with technical duplicates. The experiments have been repeated at least three times. In S7 Fig, these data for mutant CqsR are presented in comparison to the strain containing WT CqsR using a Y-axis scale that improves the visibility of the dose-response curves. In S8 Fig, biological replicates with technical triplicates are shown.
Fig 7.
CqsRp-ethanolamine and CqsRp-D198N are monomeric in solution.
Size exclusion chromatography of CqsRp-ethanolamine and CqsRp-D198N, with peak volumes indicated. CqsRp-ethanolamine (MWtheor = 24.3 kD, MWexp = 22.8 kD). CqsRp-D198N (MWtheor = 24.2 kD, MWexp = 19.3 kD). Vertical lines above the absorbance traces indicate protein size standards elution volumes (peak positions). Absorbance was measured at 280 nm and reported as milliabsorbance units (mAU).
Fig 8.
Ethanolamine-induced CqsRp conformational changes.
Two different views of apo CqsRp-D198N (gray cartoon) and CqsRp-ethanolamine (magenta cartoon) aligned using their α1 helices (residues 44-77). Ethanolamine binding to the membrane distal Cache domain triggers its compaction, and the residues corresponding to helices α3 and α4 become ordered. These conformational changes in the membrane distal Cache domain result in the anticlockwise rotation of the membrane proximal Cache domain, which is more apparent in S1 Movie. Ethanolamine is represented as spheres.
Fig 9.
Cache domain binding modes for 2-amino alcohols, amino acids, and quaternary amines.
(A) The CqsRp-ethanolamine binding site, highlighting the hot-spot interaction between conserved D198 and the ethanolamine amine and hydroxyl. CqsRp is represented as blue sticks, ethanolamine is represented as a ball-and-stick model. (B) The Mlp24-glycine binding site, highlighting the hot-spot interaction between conserved D172 and the glycine amide as well as conserved R125 and the glycine carboxyl group. Mlp24 is represented as yellow sticks, glycine is represented as a ball-and-stick model. PDB: 6IOQ [63]. (C) The PctD-acetylcholine binding site. Quaternary amines adopt orientations driven by cation-pi bonds formed between the quaternary amine nitrogen and the side chain of an aromatic residue in the binding pocket. PctD is represented as green sticks, acetylcholine is represented as a ball-and-stick model. PDB: 7PRR [64]. Bonds noted in the discussion are depicted as black dashed lines.