Figure 1.
Schematic of LuxI/LuxR regulation of V.fischeri bioluminescence, and bulk response.
(A) LuxI synthesizes the autoinducer AI (N-3-oxohexanoyl-L-homoserine lactone) which binds to LuxR, the transcriptional activator for the luminescence genes luxCDABE [6]; (B) Luminescence response of a bulk culture of V.fischeri strain MJ11 growing in defined medium at room temperature. The points show the response of a (bulk) population of exponential phase cells in a 48-well plate, following addition of exogenous autoinducer (AI) at time t = 0. After 70 minutes an AI-dependent response is developing. After 130 minutes the response has reached a steady state. Data for t≥130 minutes are fit to a cooperative binding model (black dotted curve) to give an equilibrium constant Keq = 200±10 nM and Hill coefficient n = 2.6±0.4. Luminescence data are normalized to the optical density at 600 nm to give the luminescence per cell, in arbitrary units.
Figure 2.
Individual V.fischeri imaged in dark field and bioluminescence.
(A) Dark field (externally illuminated) and (B) bioluminescence (light emission) images of V.fischeri cells adhered to the glass window of the perfusion chamber at 24°C in the presence of 500 nM exogenous AI. The cells appear as rods (∼3–5 µm long) in the dark field image. The bioluminescence image shows in false color the luminescent emission detected in a 16 minute exposure. Images were collected in an inverted microscopy configuration with an intensified CCD camera and a 100× oil immersion objective.
Figure 3.
Luminescence of individual V.fischeri cells following addition of autoinducer, and detection stability test.
At each autoinducer concentration, roughly 25–40 MJ11 cells were imaged repeatedly over a period of ∼4 hrs following introduction (at t = 0) of exogenous autoinducer AI at the indicated concentration. The light emission from each cell was quantified through analysis of a series of 10-minute camera exposures (see Materials & Methods). The state of induction of the initial cell culture determines the luminescence of the cells at t = 0. However, once adhered in the flow chamber and exposed to the flow of medium (containing exogenous AI), the cells respond by adjusting their luminescent output. This leads to a transient increase or decrease in the emission over the next ∼1–2 hrs. After ∼3 hrs the cells have adapted to the applied AI level. The control shows an experimental verification of the stability and sensitivity of microscopy and data analysis. For this measurement, green fluorescent latex spheres were illuminated with a severely attenuated blue light source and then imaged with the same camera settings, magnification, 10-minute exposure time, and data analysis, as used for the V.fischeri measurements. Image focus and excitation intensity were not adjusted during the 4 hr measurement. Twelve representative trajectories are shown. See Materials and Methods and Text S1. The time-dependence of all emission versus time “trajectories” in this figure has been smoothed by a Gaussian filter with width σ = 10 minutes.
Figure 4.
Spreading of the luminescence histogram over time.
(A) Cell brightness histograms for MJ11 cells at the indicated times, following introduction of 1000 nM AI at t = 0. (B) Median (red curve) cell brightness and the 25% and 75% percentiles of brightness (blue curves). The distribution of intensities broadens as the cells response to the exogenous AI signal. A substantial fraction of the cells emit near the detection threshold (∼10–20 photons/minute/cell) even at t = 4 hrs.
Figure 5.
Histograms of luminescence levels and onset times at high autoinducer concentration.
(A) Distribution of luminescence levels detected for individual V.fischeri cells, at time t = 240 minutes after autoinducer (AI, 3OC6HSL) was introduced at concentrations indicated. Cells emitting ∼10–20 photons/minute are at the measurement uncertainty, i.e. are consistent with no emission. (B) Distribution of luminescence onset times t1/2 in the presence of 200 nM and 1000 nM AI. The onset time t1/2 is the time at which the luminescence output I(t) of a particular cell is halfway between its initial value I(t = 0) and its final value I(t≈250 minutes), when AI was introduced at t = 0.
Figure 6.
Variation and mean of luminescence levels versus autoinducer concentration.
(A) Coefficient of variation (cv = standard deviation/mean) in the luminescence of different cells. Variation is calculated from emission levels recorded t >100 minutes after introduction of exogenous AI; (B) Luminescence emission detected from 188 individual cells (blue circles) after 150–250 minutes exposure to AI. Data for each AI concentration represents a different group of cells. Solid curve (blue) is a fit to a cooperative binding model, giving Keq≈120±20 nM and Hill coefficient n≈2.7±0.8. For comparison with the expected average behavior, the dashed curve (red) shows the AI response that is obtained from a bulk population after 150–250 minutes in autoinducer (Figure 1).
Figure 7.
Heterogeneity of native luminescence versus fluorescence reporter for V.fischeri quorum system.
(A) Histogram of bioluminescence emission levels from 47 individual V.fischeri cells of wildtype strain MJ11, following induction by 1000 nM AI. The luminescence levels are normalized to the median value. (B) Histogram of fluorescence levels for 127 individual V.fischeri cells of mutant JB10, following induction by 1000 nM AI. The JB10 mutant contains a chromosomal gfp insertion between luxI and luxC in the LuxI/LuxR system. Fluorescence values are normalized to the median value. Both luminescence and fluorescence reporters for the QS system show a broadly heterogeneous response at full induction, although the fluorescence shows slightly less variability (cv≈0.8) than the luminescence (cv≈1.0).