In vitro reconstitution of the CqsS → LuxU → LuxO phosphorylation cascade. A. Auto-phosphorylation of CqsS and phosphotransfer to LuxU and to LuxO were assayed with wild-type CqsS, CqsS His^-, CqsS Cat^- and CqsS Asp^-. All phosphorylated proteins were run on the same gel and extracted. Only the relevant regions are shown for simplicity. B. A phosphorylation reaction was carried out with CqsS, LuxU and LuxO for 1 min and divided in half. One half was supplemented with DMSO (left) and the other with 500 µM CAI-1 (right). Samples were taken at the indicated time points. The top, middle and bottom rows show the phosphorylated CqsS, LuxU and LuxO proteins respectively. C. Experiments in (B) were performed in duplicate. Band intensities for CqsS∼P (circles), LuxU∼P (triangles) and LuxO∼P (squares) when DMSO was added (open symbols) and when CAI-1 was added (closed symbols) were normalized to the time zero level of each protein.

CqsS antagonism occurs in vitro. Antagonism was examined with the CqsS wild-type receptor (left and right) and the CqsS^C170Y mutant receptor (middle). Reactions containing LuxU and either wild-type CqsS or CqsS^C170Y were supplemented with the ligand(s) indicated above each lane. Agonists were present at 10 µM while antagonists were present at 100 µM (left and middle) or 500 µM (right). Reactions were carried out for 30 s. Both phosphorylated proteins were run on the same gel and extracted. Only the relevant regions are shown for simplicity. B. Experiments in (A) were performed in duplicate and band intensities were normalized to the respective DMSO control in each set of experiments. White bars and black bars show the quantification for CqsS and LuxU respectively.

Phosphotransfer steps subsequent to CqsS auto-phosphorylation are not CAI-1 regulated. Auto-phosphorylation of His194 on CqsS Asp^- and phosphotransfer from wild-type CqsS to LuxU were measured in the presence of various concentrations of CAI-1. Reactions were carried out for 30 s. Experiments were performed in duplicate and band intensities of phosphorylated CqsS Asp^- (white bars) and LuxU (black bars) were quantified and normalized to their respective DMSO controls.

CAI-1 affects dephosphorylation of CqsS His∼P by ADP. 100 µM ADP was added to phosphorylated CqsS Asp^- together with DMSO (open circles) or 500 µM CAI-1 (closed circles). At that time of ADP and ligand addition, 1 mM ATP was also added to terminate new labelling. Aliquots were removed at the indicated time points. The same experiment was performed with 100 µM EDTA present together with DMSO (open squares) or CAI-1 (closed squares). Experiments were performed in duplicate and band intensities were normalized to the time zero band from each experiment.

The V. cholerae CqsS/CAI-1 quorum-sensing phosphorelay system. CqsA synthesizes CAI-1, which is (S)-3-hydroxytridecan-4-one and CqsS is the CAI-1 receptor. At low cell density (LCD, left), when CAI-1 concentration is below the detection limit, CqsS functions as a kinase. Following auto-phosphorylation at His194, the phosphoryl group is transferred to Asp618 on the CqsS receiver domain. The next transfer is to His58 on LuxU. LuxU, in turn, transfers the phosphoryl group to Asp47 on LuxO. Once phosphorylated, LuxO activates the transcription of genes encoding four small regulatory RNAs called Qrr1-4. Qrr1-4 activates the translation of AphA and represses the translation of HapR. AphA regulates genes that are beneficial for individual behaviours. At high cell density (HCD, right), CAI-1 accumulates, binds CqsS, and switches CqsS to a phosphatase. Phospho-flow is reversed and LuxO is dephosphorylated. Qrr1-4 are not produced, and HapR protein is translated. HapR regulates genes that promote group behaviours. Open arrows denote phospho-flow and closed arrows denote gene regulation and CAI-1 synthesis. We note that the trans phosphorylation of CqsS His194 is shown for simplicity based on data from other TCS systems. We do not exclude the possibility of cis phosphorylation. In this study, the reverse phosphate flow from LuxU to CqsS that occurs at high cell density is termed the CqsS phosphatase activity for continuity with previous reports. We note that the canonical definition of phosphatase activity in a two-component system refers to that of a histidine kinase targeting the aspartyl-phosphate on the partner response regulator.

Receptor-ligand specificities are maintained in vitro. A. CqsS and LuxU phosphorylation were examined in the presence of DMSO or 100 µM CAI-1, Decanoic acid, HAI-1 (3-hydroxy-C4-HSL), or AI-2 [(2S,4S)-2-methyl-2,3,4-tetrahydroxytetrahydrofuran-borate]. Both phosphorylated proteins were run on the same gel and extracted. Only the relevant regions are shown for simplicity. B. Structures of CAI-1, C8-CAI-1 and P-CAI-1. C. CqsS auto-phosphorylation and phosphotransfer to LuxU were examined with the wild-type CqsS receptor (left), the CqsS^C170Y (middle) and the CqsS^F162A (right) mutant receptors in the presence of DMSO or 100 µM of CAI-1, C8-CAI-1 or P-CAI-1. Reactions were carried out for 30 s. Both phosphorylated proteins were run on the same gel and extracted. Only the relevant regions are shown for simplicity. D. Experiments in (C) were performed in duplicate and band intensities were normalized to the respective DMSO control in each set of experiments. White bars and black bars show the quantification for CqsS and LuxU respectively.

CAI-1 regulates CqsS auto-phosphorylation. A. CqsS auto-phosphorylation was examined using the CqsS Asp^- mutant construct in the presence of DMSO (left) or 500 µM CAI-1 (right). Samples were taken at the indicated time points. B. Experiments in (A) were performed in duplicate. Band intensities from the DMSO control (circles) and CAI-1 treated (squares) samples were quantified and normalized to the 65 s time point band from the DMSO control in each experiment.

The CqsS phosphatase activity is not regulated by CAI-1. Phosphorylated LuxU was purified and divided in half. DMSO (circles) or 500 µM CAI-1 (squares) was added to one half of the sample. CqsS was added to both reaction mixtures at time zero and aliquots were subsequently removed at the indicated time points. Experiments were performed in duplicate and band intensities were normalized to the time zero band from each experiment.