Ion channels enable electrical communication in bacterial communities

Journal name:
Nature
Volume:
527,
Pages:
59–63
Date published:
DOI:
doi:10.1038/nature15709
Received
Accepted
Published online

Abstract

The study of bacterial ion channels has provided fundamental insights into the structural basis of neuronal signalling; however, the native role of ion channels in bacteria has remained elusive. Here we show that ion channels conduct long-range electrical signals within bacterial biofilm communities through spatially propagating waves of potassium. These waves result from a positive feedback loop, in which a metabolic trigger induces release of intracellular potassium, which in turn depolarizes neighbouring cells. Propagating through the biofilm, this wave of depolarization coordinates metabolic states among cells in the interior and periphery of the biofilm. Deletion of the potassium channel abolishes this response. As predicted by a mathematical model, we further show that spatial propagation can be hindered by specific genetic perturbations to potassium channel gating. Together, these results demonstrate a function for ion channels in bacterial biofilms, and provide a prokaryotic paradigm for active, long-range electrical signalling in cellular communities.

At a glance

Figures

  1. Biofilms produce synchronized oscillations in membrane potential.
    Figure 1: Biofilms produce synchronized oscillations in membrane potential.

    a, Biofilms generate collective metabolic oscillations resulting from long-range metabolic interactions between interior and peripheral cells18. It remains unclear how microscopic bacteria are capable of communicating over such macroscopic distances within biofilm communities. b, Schematic of the microfluidic device used throughout this study (left). Phase contrast image of a biofilm growing in the microfluidic device with the cell trap highlighted in yellow (right). Scale bar, 100 μm. c, Global oscillations in membrane potential, as reported by thioflavin T (ThT), within the biofilm community. ThT is positively charged but not known to be actively transported, so it can be retained in cells due to their negative membrane potential inside the cell. ThT fluorescence increases when the inside of the cell becomes more negative, and thus ThT is inversely related to the membrane potential. Scale bar, 0.15 mm. Representative images shown are taken from over 75 independent biofilms. a.u., arbitrary units. d, Membrane potential oscillations are highly synchronized even between the most distant regions of the biofilm. To analyse synchronization, the edge region of the biofilm was identified and straightened (left) then plotted over time (right). e, Time traces of the heat map shown in d. Indicated in bold is the mean of 30 traces.

  2. Potassium release is involved in active signal propagation within the biofilm.
    Figure 2: Potassium release is involved in active signal propagation within the biofilm.

    a, An extracellular fluorescent chemical dye (APG-4) reports the concentration of potassium in the media (Extended Data Fig. 2a, b). For comparison, the same cells are shown stained with ThT, which is inversely related to the membrane potential. These images depict cells at the peak of the ThT oscillation cycle. Representative images are selected from six independent experiments. Scale bar, 2 μm. b, Global oscillations in extracellular potassium throughout the biofilm. A white line indicates the edge of the biofilm. Representative images are selected from six independent experiments. Scale bar, 0.2 mm. c, Oscillations in membrane potential and extracellular potassium are synchronized, suggesting that potassium release is involved in global membrane potential oscillations. ThT is inversely related to the membrane potential. Representative traces are taken from the experiment shown in b. d, A chemical potassium clamp (300 mM KCl, matching the intracellular concentration29) prevents the formation of potassium electrochemical gradients across the cellular membrane. e, Clamping net potassium flux quenches oscillations in membrane potential. Representative trace is selected from two independent experiments. f, Illustration of the differences between passive signalling (diffusion) and active signalling. When cells passively respond to a signal, the range that the signal can propagate is limited due to the decay of signal amplitude. In contrast, when cells actively respond by amplifying the signal, propagation can extend over greater distances. g, We measured propagation of extracellular potassium by measuring APG-4 in time and along a length of approximately 1.5 mm within the biofilm. h, Extracellular potassium amplitude is relatively constant as the signal propagates, in contrast to the predicted amplitude decay of a passive signal. Representative data selected from six independent experiments. The diffusion line is calculated using the 2D diffusion equation and the diffusion coefficient for potassium within biofilms (Supplementary Information).

  3. The molecular mechanism of signal propagation involves potassium channel gating.
    Figure 3: The molecular mechanism of signal propagation involves potassium channel gating.

    a, yugO is a potassium channel in B. subtilis that is gated intracellularly by a trkA domain, which is regulated by the metabolic state of the cell34, 35, 36. Withdrawing glutamate (the sole nitrogen source in MSgg media) induces an increase in extracellular potassium (APG-4) for wild-type (WT) but not the yugO deletion strain. Error bars indicate the mean ± s.d. for three independent biofilms each. b, An external potassium shock (300 mM KCl) induces a short-term membrane potential depolarization in both wild-type and yugO deletion strains. However, in the wild type this initial depolarization was followed by hyperpolarization, which is not observed in the yugO deletion strain (mean ± s.d. for 12 traces drawn from 3 biofilms each). ThT is inversely related to the membrane potential. c, Proposed model for potassium signalling. The initial trigger for potassium release is metabolic stress caused by glutamate limitation. External potassium depolarizes neighbouring cells, producing further nitrogen limitation by limiting glutamate uptake, and thus produces further metabolic stress. This cycle results in cell–cell propagation of the potassium signal. d, A minimal conductance-based model describing the dynamics of the cell’s membrane potential in terms of a single potassium channel and a leak current. Consistent with our experimental results, this simple model exhibits transient depolarization followed by hyperpolarization in response to local increases in extracellular potassium concentration. e, The model predicts that manipulating channel gating and conductance will result in decaying amplitude in the spatial propagation of membrane potential oscillations. f, Maximum intensity projection of membrane potential change illustrating attenuated communication within the biofilm in a yugOΔtrkA deletion compared to wild-type biofilms (top). Heat map of oscillations taken from wild-type and yugOΔtrkA mutant biofilms (bottom). Representative images are taken from three independent biofilm experiments in which wild-type and yugOΔtrkA biofilms are compared head-to-head. Scale bars, 8 μm. g, Quantification of normalized pulse amplitude from wild-type (n = 8 pulses) and yugOΔtrkA (n = 12 pulses) mutant biofilms (mean ± s.e.m.).

  4. Thioflavin T (ThT) is a fluorescent reporter that is inversely related to the membrane potential.
    Extended Data Fig. 1: Thioflavin T (ThT) is a fluorescent reporter that is inversely related to the membrane potential.

    a, ThT and DiSC3(5), an established reporter of membrane potential in bacteria23, both oscillate within biofilms. ThT has an approximately three fold higher sensitivity to changes in membrane potential compared to DiSC3(5). Sensitivity is defined as the ratio between peak height and error in peak height. Error bars indicate mean ± s.d. (n = 8 biofilm regions, averaged over the 4 pulses shown). b, The cellular ThT fluorescence depends on the external pH, where higher pH results in greater membrane potential, as expected24. ThT itself is insensitive to these pH changes and the traces are background subtracted to eliminate possible artefacts. Representative trace is selected from three independent biofilms. c, Oscillations in ThT and growth rate are inversely correlated, linking membrane potential oscillations to the metabolic cycle which produces periodic growth pauses18. Growth rate is calculated by taking the derivative of biofilm radius over time (Supplementary Information). Representative trace is selected from over 75 independent biofilms. d, Replacing glutamate with 0.2% glutamine, which eliminates the need to take up glutamate or retain ammonium, quenches ThT oscillations. This further suggests that ThT oscillations are specific to the metabolic cycle involving glutamate and ammonium. A representative trace was selected from three independent experiments.

  5. A fluorescent reporter of extracellular potassium (APG-4) indicates that potassium has a role in membrane potential oscillations.
    Extended Data Fig. 2: A fluorescent reporter of extracellular potassium (APG-4) indicates that potassium has a role in membrane potential oscillations.

    a, High-resolution images showing the intracellular localization of ThT and primarily extracellular localization of APG-4 (top). Quantification of ThT and APG-4 along the 2 μm profile indicated in the phase image indicates that APG-4 does not significantly diffuse into the cell (bottom). Representative images are selected from six independent experiments. b, Induction curve for APG-4 generated using externally supplemented KCl. The experiment was repeated twice. c, Oscillations in extracellular potassium in the surrounding cell-free region during biofilm oscillations. These oscillations occurred during the experiment shown in Fig. 2b, c and the pulses are synchronized between the biofilm and the surrounding cell-free region. Representative trace is selected from six independent experiments. d, Induction curve for ANG-2 generated using externally supplemented NaCl. The experiment was repeated twice. e, Simultaneous measurement of ThT and ANG-2 indicates a lack of oscillations in extracellular sodium. Representative trace selected from three independent biofilms. f, Furthermore, perturbing extracellular sodium concentrations in the media had no detectable effect on membrane potential oscillations. A representative trace was selected from four independent experiments.

  6. Active propagation of potassium signal within the biofilm.
    Extended Data Fig. 3: Active propagation of potassium signal within the biofilm.

    a, A chemical potassium clamp (300 mM KCl, matching the intracellular concentration29, and 30 μM valinomycin) prevents the formation of potassium electrochemical gradients across the cellular membrane. Valinomycin is an antibiotic that creates potassium-specific carriers in the cellular membrane32. b, Clamping net potassium flux quenches oscillations in membrane potential. A representative trace was selected from two independent biofilms. c, Propagation of extracellular potassium is estimated by tracking the half-maximal position of the pulse over time. Representative traces are shown for a single pulse selected from one of six independent experiments. d, Propagation of extracellular potassium is relatively constant over time in contrast to diffusion that is expected to decay. The diffusion line is calculated using the mean squared displacement (MSD) and the diffusion coefficient for potassium in biofilms (Supplementary Information). Slopes are calculated from the same representative data shown in c.

  7. External potassium affects the metabolic state of the cell.
    Extended Data Fig. 4: External potassium affects the metabolic state of the cell.

    a, A potassium shock (300 mM KCl) produces an initial ThT decrease (depolarization) followed by a period of sustained ThT increase (hyperpolarization). ThT is inversely related to the membrane potential. A corresponding pulse in APG-4 during this ThT increase suggests that hyperpolarization is due to release of potassium. APG-4 signal due to the external potassium shock itself was subtracted using the cell-free background near the biofilm. A representative trace was selected from three independent experiments. b, ThT spikes in response to external potassium shock (300 mM KCl) but not an equivalent shock of 300 mM sorbitol, an uncharged solute. A representative trace was selected from three independent experiments. c, The hyperpolarization response occurs when cells are grown in glutamate but not when glutamate is replaced by 0.2% glutamine, which bypasses the need to take up glutamate or retain ammonium. A representative trace was selected from four independent biofilms.

Tables

  1. List of strains used in this study
    Extended Data Table 1: List of strains used in this study
  2. Parameter values used in the model
    Extended Data Table 2: Parameter values used in the model

Videos

  1. Video 1: Global oscillations in membrane potential (ThT) in a growing biofilm.
    Video 1: Video 1: Global oscillations in membrane potential (ThT) in a growing biofilm.
    Global oscillations in membrane potential (ThT) in a growing biofilm.
  2. Video 2: Waves of extracellular potassium (APG-4) in a growing biofilm.
    Video 2: Video 2: Waves of extracellular potassium (APG-4) in a growing biofilm.
    Waves of extracellular potassium (APG-4) in a growing biofilm.
  3. Video 3: Oscillations in membrane potential (ThT) in a wild type and yugOtrkA mutant biofilm
    Video 3: Video 3: Oscillations in membrane potential (ThT) in a wild type and yugOΔtrkA mutant biofilm
    The yugOΔtrkA mutant strain lacks the gating domain for the YugO potassium channel and is unable to signal the edge of the biofilm.

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Author information

  1. These authors contributed equally to this work.

    • Jintao Liu &
    • Munehiro Asally

Affiliations

  1. Division of Biological Sciences, University of California San Diego, California 92093, USA

    • Arthur Prindle,
    • Jintao Liu,
    • San Ly &
    • Gürol M. Süel
  2. Warwick Integrative Synthetic Biology Centre, School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK

    • Munehiro Asally
  3. Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain

    • Jordi Garcia-Ojalvo

Contributions

G.M.S., A.P., J.L., M.A. and J.G.-O. designed the research, A.P. and J.L. performed the experiments, J.L. and A.P. performed the data analysis, J.G.O. performed the mathematical modelling, S.L. made the bacteria strains, and G.M.S., A.P., J.L. and J.G.-O. wrote the manuscript. All authors discussed the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Thioflavin T (ThT) is a fluorescent reporter that is inversely related to the membrane potential. (253 KB)

    a, ThT and DiSC3(5), an established reporter of membrane potential in bacteria23, both oscillate within biofilms. ThT has an approximately three fold higher sensitivity to changes in membrane potential compared to DiSC3(5). Sensitivity is defined as the ratio between peak height and error in peak height. Error bars indicate mean ± s.d. (n = 8 biofilm regions, averaged over the 4 pulses shown). b, The cellular ThT fluorescence depends on the external pH, where higher pH results in greater membrane potential, as expected24. ThT itself is insensitive to these pH changes and the traces are background subtracted to eliminate possible artefacts. Representative trace is selected from three independent biofilms. c, Oscillations in ThT and growth rate are inversely correlated, linking membrane potential oscillations to the metabolic cycle which produces periodic growth pauses18. Growth rate is calculated by taking the derivative of biofilm radius over time (Supplementary Information). Representative trace is selected from over 75 independent biofilms. d, Replacing glutamate with 0.2% glutamine, which eliminates the need to take up glutamate or retain ammonium, quenches ThT oscillations. This further suggests that ThT oscillations are specific to the metabolic cycle involving glutamate and ammonium. A representative trace was selected from three independent experiments.

  2. Extended Data Figure 2: A fluorescent reporter of extracellular potassium (APG-4) indicates that potassium has a role in membrane potential oscillations. (357 KB)

    a, High-resolution images showing the intracellular localization of ThT and primarily extracellular localization of APG-4 (top). Quantification of ThT and APG-4 along the 2 μm profile indicated in the phase image indicates that APG-4 does not significantly diffuse into the cell (bottom). Representative images are selected from six independent experiments. b, Induction curve for APG-4 generated using externally supplemented KCl. The experiment was repeated twice. c, Oscillations in extracellular potassium in the surrounding cell-free region during biofilm oscillations. These oscillations occurred during the experiment shown in Fig. 2b, c and the pulses are synchronized between the biofilm and the surrounding cell-free region. Representative trace is selected from six independent experiments. d, Induction curve for ANG-2 generated using externally supplemented NaCl. The experiment was repeated twice. e, Simultaneous measurement of ThT and ANG-2 indicates a lack of oscillations in extracellular sodium. Representative trace selected from three independent biofilms. f, Furthermore, perturbing extracellular sodium concentrations in the media had no detectable effect on membrane potential oscillations. A representative trace was selected from four independent experiments.

  3. Extended Data Figure 3: Active propagation of potassium signal within the biofilm. (151 KB)

    a, A chemical potassium clamp (300 mM KCl, matching the intracellular concentration29, and 30 μM valinomycin) prevents the formation of potassium electrochemical gradients across the cellular membrane. Valinomycin is an antibiotic that creates potassium-specific carriers in the cellular membrane32. b, Clamping net potassium flux quenches oscillations in membrane potential. A representative trace was selected from two independent biofilms. c, Propagation of extracellular potassium is estimated by tracking the half-maximal position of the pulse over time. Representative traces are shown for a single pulse selected from one of six independent experiments. d, Propagation of extracellular potassium is relatively constant over time in contrast to diffusion that is expected to decay. The diffusion line is calculated using the mean squared displacement (MSD) and the diffusion coefficient for potassium in biofilms (Supplementary Information). Slopes are calculated from the same representative data shown in c.

  4. Extended Data Figure 4: External potassium affects the metabolic state of the cell. (94 KB)

    a, A potassium shock (300 mM KCl) produces an initial ThT decrease (depolarization) followed by a period of sustained ThT increase (hyperpolarization). ThT is inversely related to the membrane potential. A corresponding pulse in APG-4 during this ThT increase suggests that hyperpolarization is due to release of potassium. APG-4 signal due to the external potassium shock itself was subtracted using the cell-free background near the biofilm. A representative trace was selected from three independent experiments. b, ThT spikes in response to external potassium shock (300 mM KCl) but not an equivalent shock of 300 mM sorbitol, an uncharged solute. A representative trace was selected from three independent experiments. c, The hyperpolarization response occurs when cells are grown in glutamate but not when glutamate is replaced by 0.2% glutamine, which bypasses the need to take up glutamate or retain ammonium. A representative trace was selected from four independent biofilms.

Extended Data Tables

  1. Extended Data Table 1: List of strains used in this study (48 KB)
  2. Extended Data Table 2: Parameter values used in the model (236 KB)

Supplementary information

Video

  1. Video 1: Video 1: Global oscillations in membrane potential (ThT) in a growing biofilm. (1.44 MB, Download)
    Global oscillations in membrane potential (ThT) in a growing biofilm.
  2. Video 2: Video 2: Waves of extracellular potassium (APG-4) in a growing biofilm. (637 KB, Download)
    Waves of extracellular potassium (APG-4) in a growing biofilm.
  3. Video 3: Video 3: Oscillations in membrane potential (ThT) in a wild type and yugOΔtrkA mutant biofilm (349 KB, Download)
    The yugOΔtrkA mutant strain lacks the gating domain for the YugO potassium channel and is unable to signal the edge of the biofilm.

PDF files

  1. Supplementary Information (682 KB)

    This file contains Supplementary Text and Data and Supplementary References.

Additional data