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.
- 2002) & Spiking Neuron Models: Single Neurons, Populations, Plasticity (Cambridge Univ. Press,
- 2001) Ion Channels of Excitable Membranes (Sinauer Associates,
- Potassium channels and the atomic basis of selective ion conduction. Biosci. Rep. 24, 75–100 (2004)
- The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998) et al.
- A prokaryotic voltage-gated sodium channel. Science 294, 2372–2375 (2001) et al.
- A biological role for prokaryotic ClC chloride channels. Nature 419, 715–718 (2002) , , &
- Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002) et al.
- Functional characterization of a potassium-selective prokaryotic glutamate receptor. Nature 402, 817–821 (1999) , , &
- Prokaryotic K+ channels: From crystal structures to diversity. FEMS Microbiol. Rev. 29, 961–985 (2005) , , , &
- Ion channels in microbes. Methods Enzymol. 294, 507–524 (1998) , , , &
- Pressure-sensitive ion channel in Escherichia coli. Proc. Natl Acad. Sci. USA 84, 2297–2301 (1987) , , , &
- Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322 (1999) , &
- Bacterial biofilms: from the natural environment to infectious diseases. Nature Rev. Microbiol. 2, 95–108 (2004) , &
- Control of cell fate by the formation of an architecturally complex bacterial community. Genes Dev. 22, 945–953 (2008) , , &
- Localized cell death focuses mechanical forces during 3D patterning in a biofilm. Proc. Natl Acad. Sci. USA 109, 18891–18896 (2012) et al.
- Liquid transport facilitated by channels in Bacillus subtilis biofilms. Proc. Natl Acad. Sci. USA 110, 848–852 (2013) et al.
- Temporal control of self-organized pattern formation without morphogen gradients in bacteria. Mol. Syst. Biol. 9, 697 (2013) et al.
- Metabolic co-dependence gives rise to collective oscillations within microbial communities. Nature 523, 550–554 (2015) et al.
- Characterization of the proton/glutamate symport protein of Bacillus subtilis and its functional expression in Escherichia coli. J. Bacteriol. 177, 2863–2869 (1995) , , &
- AmtB-mediated NH3 transport in prokaryotes must be active and as a consequence regulation of transport by GlnK is mandatory to limit futile cycling of NH4+/NH3. FEBS Lett. 585, 23–28 (2011) et al.
- Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein. Science 333, 345–348 (2011) , , &
- Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load. Biophys. J. 93, 294–302 (2007) , , &
- Membrane potential is important for bacterial cell division. Proc. Natl Acad. Sci. USA 107, 12281–12286 (2010) &
- Molecular aspects of bacterial pH sensing and homeostasis. Nature Rev. Microbiol. 9, 330–343 (2011) , &
- The roles and regulation of potassium in bacteria. Prog. Nucleic Acid Res. Mol. Biol. 75, 293–320 (2003)
- Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis. Proc. Natl Acad. Sci. USA 106, 280–285 (2009) , , , &
- Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis. J. Bacteriol. 187, 8462–8469 (2005) , , &
- The structure of the KtrAB potassium transporter. Nature 496, 323–328 (2013) , &
- KtrAB and KtrCD: Two K+ uptake systems in Bacillus subtilis and their role in adaptation to hypertonicity. J. Bacteriol. 185, 1289–1298 (2003) , , &
- The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis. J. Gen. Microbiol. 136, 2527–2535 (1990) , &
- A novel optical intracellular imaging approach for potassium dynamics in astrocytes. PLoS ONE 9, 1–9 (2014) &
- Voltage clamp effects on bacterial chemotaxis. J. Bacteriol. 159, 605–610 (1984) &
- MstX and a putative potassium channel facilitate biofilm formation in Bacillus subtilis. PLoS ONE 8, e60993 (2013) , &
- Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature 496, 317–322 (2013) et al.
- A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch. Cell 109, 781–791 (2002) , , &
- NAD+ binding to the Escherichia coli K+-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport. Mol. Microbiol. 9, 533–543 (1993) , , , &
- Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!. Mol. Microbiol. 32, 223–232 (1999)
- Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating. J. Gen. Physiol. 117, 165–180 (2001) , &
- A quantitative description of membrane current and its applications to conduction and excitation in nerve. J. Physiol. (Lond.) 117, 500–544 (1952) &
- Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346 (2005) &
- Iron-oxide minerals affect extracellular electron-transfer paths of Geobacter spp. Microbes Environ. 28, 141–148 (2013) , &
- Filamentous bacteria transport electrons over centimetre distances. Nature 491, 218–221 (2012) et al.
- Electrical spiking in bacterial biofilms. J. R. Soc. Interface 12, 1–10 (2014) et al.
- Neurometabolism in human epilepsy. Epilepsia 49, 31–41 (2008) et al.
- Glutamate-glutamine cycling in the epileptic human hippocampus. Epilepsia 43, 703–710 (2002) , , , &
- Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J. Nutr. 130, 1007S–1015S (2000)
- A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales. Mol. Microbiol. 76, 559–575 (2010) &
- Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions. FEMS Microbiol. Lett. 206, 197–200 (2002) , , &
- 1985) Handbook of Aqueous Electrolyte Solutions: Physical Properties, Estimation and Correlation Methods (Ellis Horwood Ltd,
- Diffusion in biofilms. J. Bacteriol. 185, 1485–1491 (2003)
Extended data figures and tables
Extended Data Figures
- 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.
- 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.
- 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.
- 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
- 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.
- 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.
- 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.
- Supplementary Information (682 KB)
This file contains Supplementary Text and Data and Supplementary References.