The Vibrio harveyi quorum-sensing system uses shared regulatory components to discriminate between multiple autoinducers

Abstract

The quorum-sensing bacterium Vibrio harveyi produces and responds to three autoinducers (AIs), and this sensory information converges to control the expression of bioluminescence, biofilm formation, type III secretion (TTS), and protease production. The AIs are detected by cognate sensor histidine kinases that all relay phosphate to the shared response regulator LuxO. LuxO indirectly represses the master regulator of quorum sensing, LuxR, through the activation of multiple genes encoding small regulatory RNAs (called qrr genes for Quorum Regulatory RNA). Here we use differential fluorescence induction to identify 50 quorum-sensing-controlled promoters. Some promoters only showed significant responses in the simultaneous presence of all three AIs, while others displayed substantial responses to the individual AIs. A differential response to each AI input state was also observed for qrr and luxR expression and LuxR protein production. Individual cell analyses revealed that, in each case, all the bacteria in the population respond in unison to the various AI inputs. We propose that the V. harveyi quorum-sensing transition is not switch-like but rather operates in a graded manner, and that this signaling arrangement, which uses shared regulatory proteins, nonetheless provides V. harveyi a mechanism to respond uniquely to different AI input states.

FIGURE 1.

Model of the V. harveyi quorum-sensing system. Three sensory systems converge to control the levels of the master regulator, LuxR. See text for details. Circles, triangles, and double pentagons represent CAI-1, HAI-1, and AI-2, respectively. Arrows denote the direction of phosphate flow in the low-cell-density state.

FIGURE 2.

LuxR directly regulates quorum-sensing-controlled genes. Expression of AI-regulated promoters fused to gfp was measured in an E. coli strain harboring a vector carrying luxR (light bars) or a vector control (black bars). Error bars indicate the standard deviations.

FIGURE 3.

Differential AI-regulation of quorum-sensing-controlled genes. Fluorescence production from the promoter gfp fusions isolated in this study was measured in the absence of AI (black diamonds), or in the presence of 10 μM AI-2 (red squares), 10 μM HAI-1 (blue triangles), or 10 μM each AI-2 and HAI-1 (green circles). Each AI-regulated promoter is depicted on the X-axis with the values for fluorescence emission plotted on the Y-axis. The fluorescence of the promoters to the left of the line marked with the asterisk (*) were multiplied by a factor of 5 so that they could be visualized on the same axis. White, gray, and black arrows show examples of class 1, 2, and 3 regulation, respectively. The four values corresponding to each arrow are boxed. The black arrows below the X-axis indicate the fusions analyzed in Figure 8.

FIGURE 4.

Three classes of AI-regulated promoters. (A) Fluorescence production from three promoters, indicated on the X-axis, in response to no AI (black bars), 10 μM AI-2 (gray bars), 10 μM HAI-1 (hatched bars), or 10 μM each AI-2 and HAI-1 (white bars). Fluorescence values are expressed as the percentage measured in the absence of AIs. Error bars indicate the standard deviations. (B) Time course of fluorescence production from clone 275 fused to an unstable GFP (LVA) variant under the four AI conditions listed in A. The OD₆₀₀ of each culture is plotted on the X-axis. (Triangles) No AI; (diamonds) AI-2; (squares) HAI-1; (circles) AI-2 and HAI-1.

FIGURE 5.

CAI-1 has no influence on quorum-sensing-regulated targets. Expression of the AI-regulated promoters in the V. harveyi triple synthase mutant JMH634 (Y-axis) and the double AI synthase mutant KM413 (X-axis) is shown. Only one promoter consistently shows twofold or greater regulation (boxed, described in text). The three fusions to qrr promoters are indicated by the circles.

FIGURE 6.

AIs repress qrr expression and activate LuxR production in a graded manner. (A) Expression of a qrr2-gfp promoter (clone 250) in the presence of no AI, 10 μM AI-2, 10 μM HAI-1, or 10 μM each AI-2 and HAI-1. Error bars indicate the standard deviations. (B) Single cell analysis of qrr2-gfp expression measured by flow cytometry. (Purple trace) Background fluorescence; (green trace) no AI; (red trace) AI-2; (blue trace) HAI-1; (orange trace) both AI-2 and HAI-1. (C) Expression of a luxR–gfp translational fusion was assayed in V. harveyi CW2005 (HAI-1⁻, AI-2⁻, luxR) following exposure to no AI, 10 μM AI-2, 10 μM HAI-1, or 10 μM each AI-2 and HAI-1. Error bars indicate the standard deviations. (D) Flow cytometry results of individual cells carrying the luxR–gfp translational fusion from C. (Purple trace) Background fluorescence; (green trace) no AI; (red trace) AI-2; (blue trace) HAI-1; (orange trace) both AI-2 and HAI-1.

FIGURE 7.

Each AI induces a distinct level of bioluminescence and LuxR production. Strain KM413 (HAI-1⁻, AI-2⁻) was exposed to no AI, 10 μM AI-2, 10 μM HAI-1, or 10 μM each AI-2 and HAI-1, and subsequently bioluminescence expression (Relative Light Units: counts per minute per milliliter at OD600nm) and LuxR protein levels were determined. Protein levels from the Western blot were quantified and are expressed as a percentage of the LuxR produced in the presence of both HAI-1 and AI-2.

FIGURE 8.

Class 2 promoters have a higher affinity for LuxR than do Class 1 promoters. Surface plasmon resonance was used to examine the interaction of LuxR with two class 2 promoters (275 and 342, A,B), and one class 1 promoter (317, C). 5′-Biotin end-labeled DNA probes were amplified and attached to a BIAcore streptavidin chip. Purified LuxR at concentrations of 0 nM (dark blue), 11.9 nM (yellow), 23.75 nM (maroon), 47.5 nM (green), 95 nM (light blue), and 190 nM (red) was passed over the chip beginning at the time indicated by the solid arrow on the X-axis. At the time indicated by the open arrow, LuxR flow was terminated, and dissociation of LuxR from the DNA was determined.