Fig 1.
Simplified V. cholerae QS circuits.
(A) Two established autoinducer–receptor pairs control QS behaviors in V. cholerae. One autoinducer–receptor pair consists of CAI-1, which is synthesized by CqsA and detected by the two-component sensor-histidine kinase, CqsS. The second autoinducer–receptor pair is AI-2, produced by LuxS and detected by LuxPQ, also a two-component sensor-histidine kinase. At LCD (left), both receptors act as kinases that promote phosphorylation of the response regulator, LuxO. Phosphorylated LuxO activates expression of genes encoding regulatory RNAs called the Qrr sRNAs. The Qrr sRNAs activate production of the LCD master regulator, AphA, and repress production of the HCD master regulator, HapR. These conditions drive biofilm formation and virulence factor production. At HCD (right), the autoinducer-bound receptors act as phosphatases that strip phosphate from LuxO, resulting in AphA repression and HapR production, conditions that promote the free-swimming, planktonic lifestyle and repression of virulence. (B) Two additional QS receptors, VpsS and CqsR, also funnel information into LuxO. Their cognate autoinducers and autoinducer synthases are not known. (C) A recently discovered QS pathway consists of the autoinducer DPO, synthesized by Tdh, and its partner receptor VqmA. At HCD, DPO-bound VqmA activates expression of a gene encoding a sRNA called VqmR. VqmR represses biofilm formation. AI-2, autoinducer-2; CAI-1, cholerae autoinducer-1; DPO, 3,5-dimethylpyrazin-2-ol; HCD, high cell density; LCD, low cell density; QS, quorum sensing; sRNA, small regulatory RNA; Tdh, threonine dehydrogenase.
Fig 2.
V. cholerae biofilm formation and dispersal under static growth conditions.
(A) Time course of a representative WT V. cholerae biofilm lifecycle as imaged by brightfield microscopy using high magnification (63× objective). (B) Left panels: brightfield projections of V. cholerae biofilms in the indicated strains after 9 h of growth at 30°C, imaged using low-magnification (10× objective). Right panel: quantitation of V. cholerae WT and ΔvpsL biofilm biomass over time. (C) As in B for V. cholerae WT and QS mutants locked in LCD (luxO D61E) and HCD (luxO D61A) modes. (D) As in B for V. cholerae WT and the LCD-locked ΔhapR strain. (E) As in B for V. cholerae WT and the ΔvqmR strain. Data are represented as means normalized to the peak biofilm biomass of the WT strain in each experiment. In all cases, n = 3 biological and n = 3 technical replicates, ± SD (shaded). Numerical data are available in S1 Data. a.u., arbitrary unit; HCD, high cell density; LCD, low cell density; QS, quorum sensing; WT, wild type.
Fig 3.
AphA and HapR abundances vary inversely during biofilm formation.
(A) Representative image series showing the formation of an individual biofilm harboring the constitutive reporter PTAC-mRuby3 and AphA-mNG. (B) Quantitation of the AphA-mNG fluorescence (black line) relative to the control mRuby3 fluorescence (magenta line) over the course of biofilm development. n = 24 biofilms from three biological replicates. (C and D) As in A and B, respectively, for HapR-mNG. Inset in (D) represents the AphA-mNG/HapR-mNG ratio over time. Shading in B and D represents SD. Numerical data are available in S1 Data. a.u., arbitrary unit; mNG, mNeonGreen.
Fig 4.
Exogenous AI-2 represses WT V. cholerae biofilm formation, but MimicCAI-1 does not.
(A) Representative projections of WT V. cholerae treated with 0.25% DMSO (Ctrl), 5 μM MimicCAI-1, or 5 μM AI-2 after 9 h of biofilm growth at 30°C. (B) Quantitation of biofilm biomass for WT V. cholerae treated with 0.25% DMSO (Ctrl), 5 μM MimicCAI-1, or 5 μM AI-2 over time. Data are represented as means normalized to the peak biofilm biomass of the DMSO Ctrl. In all cases, n = 3 biological and n = 3 technical replicates, ± SD (shaded). (C) As in B for the V. cholerae luxO D61E strain treated with 0.25% DMSO (Ctrl) or 5 μM AI-2. (D) Representative images of WT V. cholerae producing HapR-mNG after treatment as in B. (E) Quantitation of HapR-mNG signal relative to the control, PTAC-mRuby3 signal over the course of biofilm development following treatment as in B. n = 24 biofilms from three biological replicates. Data are normalized to the initial intensity of the sample to which DMSO was added. (F) Representative western blot for TcpA-3×FLAG in WT V. cholerae treated with 0.25% DMSO (Ctrl), 5 μM MimicCAI-1, or 5 μM AI-2. RpoA was used as the loading control. Quantification represents three biological replicates for each condition. Values were normalized to the Ctrl. Numerical data are available in S1 Data. AI-2, autoinducer-2; a.u., arbitrary unit; CAI-1 cholerae autoinducer-1; Ctrl, control; mNG, mNeonGreen; WT, wild type.
Fig 5.
LuxPQ but not CqsS drives LCD QS behaviors.
(A) Left panel: schematic representing a V. cholerae strain that contains both QS circuits and strains that produce and detect only a single autoinducer. Middle panel: Quantitation of biofilm biomass over time for the strain possessing both QS circuits (AI-2S+R+, CAI-1S+R+; blue), only the AI-2 QS circuit (AI-2S+R+ red), and only the CAI-1 QS circuit (CAI-1S+R+; green). Right panel: the corresponding lux patterns for the strains in the middle panel. The additional black curve shows the result for the V. cholerae strain lacking all four QS receptors (ΔvpsS, ΔcqsR, ΔluxQ, ΔcqsS). (B) Left panel: representative western blot for a strain containing CqsS-3×FLAG and LuxQ-3×FLAG produced from their native loci (WT) and for a strain in which their genomic positions had been exchanged (SWAP). RpoA was used as the loading control. Quantification of the LuxQ/CqsS ratio is based on three biological replicates for each condition. Right panel: schematic showing exchange of the cqsS and luxPQ genomic locations. (C) Quantitation of biofilm biomass for the strain with the exchanged LuxPQ and CqsS alleles (CAI-1S+RSWAP, AI-2S+RSWAP) treated with 0.25% DMSO (Ctrl), 5 μM MimicCAI-1, or 5 μM AI-2 over time. (D) Left panel: schematic representing a V. cholerae strain that contains both QS circuits and strains that produce and detect only a single autoinducer in which the receptor genes are expressed from the exchanged loci. Middle panel: quantification of biofilm biomass over time for a V. cholerae strain possessing both QS circuits (CAI-1S+R+, AI-2S+R+; blue), the AI-2 circuit only, with luxPQ expressed from the cqsS locus (AI-2S+RSWAP; red), and the CAI-1 circuit only, with cqsS expressed from the luxPQ locus (CAI-1S+RSWAP; green). Right panel: the corresponding lux patterns for the strains in the middle panel. (E) Left panel: representative western blot showing CqsS-3×FLAG levels in the V. cholerae CAI-1S+R+ and CAI-1S−R+ strains. Quantification is based on three biological replicates for each condition. Middle panel: quantitation of biofilm biomass over time for the V. cholerae CAI-1S+R+, AI-2S+R+ (blue circles, blue line) and CAI-1 S−R+ (open circles, green line) strains. Right panel: The corresponding lux patterns for the strains in the middle panel. In all biofilm measurements, data are represented as means normalized to the peak biofilm biomass of the CAI-1S+R+, AI-2S+R+ strain and n = 3 biological and n = 3 technical replicates, ± SD (shaded). In all lux experiments, RLUs are defined as light production (a.u.) divided by OD600 and n = 3 biological replicates, and error bars represent SD. Numerical data are available in S1 Data. AI-2, autoinducer-2; a.u., arbitrary unit; CAI-1, cholerae autoinducer-1; Ctrl, control; LCD, low cell density; OD, optical density; QS, quorum sensing; RLU, relative light unit; WT, wild type.
Fig 6.
The V. cholerae QS circuit is a coincidence detector.
(A) Left panel: schematic for strains used in the right panel, which shows the lux expression patterns. The strains are CAI-1S+R+, AI-2S+R+ (blue), CAI-1S+R+, AI-2S−R+ (green), and CAI-1S−R+, AI-2S+R+ (red). RLUs are defined as light production (a.u.) divided by OD600. n = 3 biological replicates, and error bars represent SD. (B) Quantitation of biofilm biomass for the CAI-1S−R+, AI-2S+R+ strain to which DMSO solvent (red circles, Ctrl), 5 μM AI-2 (white circles), or 5 μM AI-2 and 5 μM MimicCAI-1 (black circles) was added. Data are represented as means normalized to the peak biofilm biomass of the control, and n = 3 biological and n = 3 technical replicates, ± SD (shaded). AI-2, autoinducer-2; a.u., arbitrary unit; CAI-1, cholerae autoinducer-1; Ctrl, control; OD, optical density; QS, quorum sensing; RLU, relative light unit.
Fig 7.
The CqsS/CAI-1 circuit is primarily a QS circuit, not a self-sensing circuit.
(A) Schematic showing self-sensing and QS. See text for details. (B) Predicted HCD gene expression level (shades of blue) over increasing cell density for cocultured secrete-and-sense and sense-only strains if a circuit exhibits exclusive self-sensing behavior (top), QS behavior (middle), or an intermediate state in which both self-sensing and QS occur (bottom). (C) Left panel: average individual cell HapR-mNG fluorescence for the V. cholerae AI-2 S+R+ (red) and the AI-2 S−R+ (black) strains grown in monoculture. Right panel: the same strains grown in coculture. (D) Left panel: average individual cell HapR-mNG fluorescence for the V. cholerae CAI-1 S+R+ (green) and the CAI-1 S−R+ (black) strains grown in monoculture. Right panel: the same strains grown in coculture. Error bars represent SD of individual cell measurements at each time point. Numerical data are available in S1 Data. AI-2, autoinducer-2; a.u., arbitrary unit; CAI-1, cholerae autoinducer-1; mNG, mNeonGreen; QS, quorum sensing.
Fig 8.
Asymmetric autoinducer thresholds drive distinct intragenus and interspecies QS responses.
(A) CAI-1 produced by V. cholerae engages its cognate CqsS receptor at very low cell densities. In contrast, AI-2 does not accumulate to sufficient levels to engage its cognate LuxPQ receptor until much higher cell densities. (B) The consequence of asymmetric receptor occupancy coupled with the QS system functioning as a coincidence detector is that AI-2 sets the pace at which QS occurs. In V. cholerae monoculture (top), the absence of AI-2 at LCD is required for biofilm formation. Thus, exogenous AI-2, such as that provided in mixed-species communities by bacteria that possess LuxS, presumably represses V. cholerae biofilm development and/or promotes dispersal (bottom). AI-2, autoinducer-2; CAI-1, cholerae autoinducer-1; LCD, low cell density; QS, quorum sensing.