Spatially propagating activation of quorum sensing in Vibrio fischeri and the transition to low population density

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Spatially propagating activation of quorum sensing in Vibrio fischeri and the transition to low population density

Keval Patel, Coralis Rodriguez, Eric V. Stabb, and Stephen J. Hagen

Phys. Rev. E 101, 062421 – Published 26 June 2020

Abstract

Bacteria communicate by secreting and detecting diffusible small molecule signals or pheromones. Using the local concentrations of these signals to regulate gene expression, individual cells can synchronize changes in phenotype population-wide, a behavior known as quorum sensing (QS). In unstirred media, the interplay between diffusion of signals, bacterial growth, and regulatory feedback can generate complex spatial and temporal patterns of expression of QS-controlled genes. Here we identify the parameters that allow a local signal to trigger a self-sustaining, traveling activation of QS behavior. Using the natural bioluminescence of wild-type Vibrio fischeri as a readout of its lux QS system, we measure the induction of a spreading QS response by a localized triggering stimulus in unstirred media. Our data show that a QS response propagates outward, sustained by positive feedback in synthesis of the diffusible signal, and that this response occurs only if the triggering stimulus exceeds a critical threshold. We also test how the autonomous or untriggered activation of the V. fischeri QS pathway changes at very low initial population densities. At the lowest population densities, clusters of cells do not transition to a self-sensing behavior, but rather remain in communication via signal diffusion until they reach sufficiently large size that their own growth slows. Our data, which are reproduced by simple growth and diffusion simulations, indicate that in part owing to bacterial growth behavior, natural QS systems can be characterized by long distance communication through signal diffusion even in very heterogeneous and spatially dispersed populations.

Received 2 February 2020
Accepted 5 May 2020

DOI:https://doi.org/10.1103/PhysRevE.101.062421

Physics Subject Headings (PhySH)

Cell communication Diffusion

Physics of Living Systems

Authors & Affiliations

Keval Patel ¹, Coralis Rodriguez², Eric V. Stabb^2,3, and Stephen J. Hagen ^1,*

¹Physics Department, University of Florida, Gainesville, Florida 32611-8440, USA
²Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA
³Department of Biological Sciences, University of Illinois, Chicago, Illinois 60607, USA

^*sjhagen@ufl.edu

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Vol. 101, Iss. 6 — June 2020

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Images

Figure 1
Autonomous activation of quorum-regulated bioluminescence in MJ11 V. fischeri embedded in semisolid medium (c-SWTO $+$ 1% agarose). (a) Bioluminescence imaged at initial population densities $n_{0}$ of $1.4 \times 10^{7}$ (top row), $1.4 \times 10^{3}$ (middle), and 14 (bottom row) cfu ${cm}^{- 3}$ at different times as indicated. (b) Luminescence emission from the plates develops at successively later times as the initial $n_{0}$ (values indicated) is decreased. (c) Pixel-to-pixel variance in the luminescence images rises sharply for plates prepared with $n_{0} < 10^{4}$ cfu ${cm}^{- 3}$ , indicating increasing spatial heterogeneity where large cell clusters produce most of the luminescence.
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Figure 2
Time $t_{a}$ to bioluminescence activation versus initial culture density $n_{0}$ . The dashed line represents $n_{0} \propto exp - k t_{a}$ [Eq. (4)] with $k = 2.8 \times 10^{- 4} s^{- 1}$ and $k C_{1} / γ^{'} = 7.5 \times 10^{8}$ cfu ${cm}^{- 3}$ . Inset: Diffusion timescale $t_{D} ≃ 1 / D n_{0}^{2 / 3}$ (black) exceeds $t_{a}$ [Eq. (4), red] for $n_{0} < 10$ .
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Figure 3
Spreading bioluminescence of V. fischeri, induced by the addition of 50 pmol exogenous 3OC6-HSL at $n_{0} = 1.4 \times 10^{6}$ cfu ${cm}^{- 3}$ . (a) Bioluminescence of strain CR5 after a 3OC6-HSL droplet is deposited at the plate center $r = 0$ at $t = 0$ . (b) Time-lapse images of plates containing strains that possess (CR5, first and second rows) or lack (CR6, third and fourth rows) a functional copy of the gene (luxI) encoding the 3OC6-HSL synthase. In the second and fourth rows, a droplet of 3OC6-HSL was added at the plate center at $t = 0$ . In the first and third rows, no 3OC6-HSL was added. (c) Bioluminescence intensity of CR5 (left) and CR6 (right) versus radial distance $r$ and time $t$ from the 3OC6-HSL droplet (bottom row: droplet added; top row: no droplet).
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Figure 4
Threshold behavior of the MJ11 response to exogenously added 3OC6-HSL. Strain MJ11 at $n_{0} = 1.4 \times 10^{4}$ cfu ${cm}^{- 3}$ in DMM is supplied a droplet of 3OC6-HSL at the plate center. (a) Image of plates at $t = 26$ h for different amounts (given in pmol) of 3OC6-HSL stimulus. (b) The luminescence intensity (luminescence counts/ ${mm}^{2}$ ) at the plate center in response to different 3OC6-HSL stimulus amounts. (c) The luminescence intensity (luminescence counts/ ${mm}^{2}$ ) at the plate center in response to different C8-HSL stimulus amounts. (d) Peak luminescence brightness versus the size of 3OC6-HSL stimulus at high and low $n_{0}$ .
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Figure 5
Effect of initial population density $n_{0}$ , growth medium, and added 3OC6-HSL on the time of response for strain MJ11. Data for growth in defined medium (DMM–agarose) and complex medium (c-SWTO–agarose) are shown. (a) Dependence of activation time $t_{a}$ on the amount of 3OC6-HSL added at $r = 0, t = 0$ for MJ11 growing in DMM–agarose and c-SWTO–agarose at high and low $n_{0}$ . (b) Threshold response of bioluminescence in DMM–agarose (left column) and c-SWTO–agarose (right column) at $n_{0} = 1.4 \times 10^{6}$ cfu ${cm}^{- 3}$ . The amount of 3OC6-HSL added at $r = 0, t = 0$ was (from top to bottom) 0, 0.2, 2, 20, 120 pmol. (c) Intensity difference $I (r, t) - I (2 cm, t)$ for MJ11 in c-SWTO, where the bioluminescence far from the droplet is subtracted from each data set in the right column of (b). The white dashed box in the bottom right panel of (b) indicates the region subtracted in (c).
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Figure 6
Simulation of quorum sensing activation of bacteria dispersed in an immobilizing medium. Bacteria secrete diffusible signal [Eq. (2)] during growth [Eq. (1)]. Parameters are given in Sec. 2. (a) At high initial population density $n_{0} > 10^{7}$ cells ${cm}^{- 3}$ , the self-activation time $t_{a}$ for the culture (in the absence of exogenously added signal) varies as $t_{a} \propto - log n_{0}$ . Behavior of $t_{a}$ at $n_{0} < 10^{7}$ cells ${cm}^{- 3}$ depends on how the late exponential phase of growth is modeled. In an average $n$ limiting model, where growth is limited by the spatial-averaged population density, $t_{a}$ approaches a constant as individual clusters self-activate. However, $t_{a}$ diverges in a local $N$ limiting model, where the rate of growth (and signal synthesis) of an isolated cluster declines as that cluster becomes large. (b) Activation threshold for the quorum circuit by exogenously added signal. Using $n_{0} = 10^{7}$ cells ${cm}^{- 3}$ , different amounts of HSL signal are initially deposited at $r = 0$ ; the fraction of cells activated is shown versus the spatially averaged population density $n$ during growth. Activated cells are those for which the local signal concentration exceeds the quorum threshold $[C (r, t) > C_{1}]$ in Eq. (2). Individual curves are simulations for 20 different quantities of exogenous HSL, logarithmically spaced from $3 \times 10^{- 14}$ to $3 \times 10^{- 11}$ mole, as indicated by the color bar. (c) Effects of doubling time $t_{d} = ln (2) / k$ , signal activation threshold $C_{1}$ , and initial population density $n_{0}$ on the fraction of the population that is at quorum when $n = 9 \times 10^{7} {cm}^{- 3}$ . The simulation parameters ( $t_{d} = 1$ h, $C_{1} = 10$ nM, $n_{0} = 10^{7} {cm}^{- 3}$ ) used in the first simulation (diamond symbols) are modified in either $t_{d}, C_{1}$ , or $n_{0}$ , as indicated for the subsequent simulations (upright and inverted triangles, and circles, respectively).
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