Figures and data
Single cell DH5α E. coli exhibit membrane potential dynamics in response to 440 nm blue light stress.
(A) Image of a sparse single cell containing ThT imaged in the microfluidic device (Scale bar: 10 μm). (B) Normalized fluorescence intensities of ion-transients for sparse cells (n = 206) as a function of time after stimulation. Each curve describes a single cell. The curve depicting the mean membrane potential dynamics is shown in black. (C) Representative image of microclustered cells containing ThT in the microfluidic device. (D) Fluorescent intensity of ion-transients for cells in microclusters as a function of time after stimulation. Each curve describes a single cell. The curve depicting the mean membrane potential dynamics is shown in black. (E) Time to first spike histogram for sparse cells (n = 206, Sparse cells in orange) and cells in microclusters (n=272, microclustered cells in blue, cells recovered from 15 clusters). The number of spiking events is shown as a function of time to the first spike. (F) Growth curves (in a semi-log coordinates) for E. coli (measured via OD600) as a function of time in the presence and absence of ThT. All data were from at least three experimental replicates. Light stress was applied for 60 minutes. The scale bars for all the images are 10 μm.
Synchronized ion-channel mediated wavefronts in E. coli biofilm:
(A) Representative fluorescence microscopy image as a function of time (1-62 min). Robust global wavefronts can be seen in an E. coli biofilm with ThT. The scale bars for all the images are 10 μm. (B) Global averaged intensity wavefront trace obtained from a 2D section of a biofilm as a function of time (mean ± SD for 30 biofilms from at least 3 experiments). (C) Globally averaged ion-channel mediated dynamics in E. coli biofilms for different sized biofilms (68-277 μm). ThT intensity is shown as a function of time.
Voltage-gated Kch potassium channel mediates ion channel membrane potential dynamics in E. coli.
(A)Schematic diagram showing the deletion of the voltage-gated Kch channel in E. coli. (B) ThT fluorescence shown as a function of time of irradiation. Deletion of Kch inactivates the second peak in single cell E. coli DH5α. Data is a mean from 52 single cells from three experimental replicates per time point for DH5α Δkch mutant (black) plotted against the wildtype single cell E. coli DH5α (blue). (C) Deletion of kch also inactivates the second peak in E. coli biofilms. Data is shown for global membrane potential dynamics for biofilms grown from E. coli DH5α Δkch mutant (black) and wildtype DH5α (blue).
Blue light influences ion-channel mediated membrane potential events in E. coli.
(A) ThT intensity as a function of time when irradiated with different powers of 440 nm light. The time to the second excitation peak is dependent on the power. All subsequent experiments were done at the irradiance value of 15.99 μW/mm2.(B)Time to first spike plotted as a function of irradiance. Blue-light irradiance affects the time to the first peak in E. coli biofilm. (C) Measurement of extracellular potassium changes for regions close to biofilms as a function of time using fluorescence microscopy. (D) LiveDead Assay using the accumulation of propidium iodide in cells(1)DH5α (n= 1842) (2) DH5α Δkch mutant (n= 1008). (E) Comparison between PI positive cells for the DH5α and the DH5α Δkch mutant. Statistical significance was calculated using the Student’s t-test. * p ≤ 0.05. (F) ThT fluorescence intensity as a function of time for cells in the presence of a ROS scavenger. E. coli cells employ ion-channel mediated dynamics to manage ROS-induced stress linked to light irradiation. Data was obtained from not less than three experiments.
Model of ion-channel mediated membrane potential in E. coli, predictions and experimental validation.
(A) Schematic diagram of the conductance model and its predictions. The model consists of two ion-channel gates. The first channel (bronze, Q) is unknown. The second channel is the potassium channel, Kch (yellow). At the onset regime 0, both ion channels are closed. Exposure to light stress results in a rapid opening of the Q channel, which has a faster-opening gating variable than the Kch channel (regime I). The Q channel has little contribution to the repolarization event, hence the overlap of regimes I and II. (B) In the Hodgkin Huxley type conductance model the current changes are modulated by the two ion channels (Q and Kch) and the leakage channel (L). (C) The predicted ThT fluorescence intensity as a function of time for the Hodgkin Huxley model. Our Hodgkin Huxley model correctly reproduces the E. coli membrane potential dynamics for the wildtype (blue) and kch-mutants (black). The wildtype has two hyperpolarization events. (D) Fluorescence intensity from our microscopy experiments with ThT as a function of time for the wildtype (blue) and Kch-mutants (black).
Role of mechananosensitive channels in the first hyperpolarization event in E. coli.
(A) A generic diagram for the membrane voltage during neuronal habituation to a constant stimulus e.g light stress31,32. (B) An illustrative diagram of membrane potential dynamics of our experiment as a function of time which is a mirror image of the ThT dynamics for comparison with (A). (C) Membrane potential dynamics for MS mutants of the wildtype, E. coli strain BW25113. (D) Membrane potential dynamics for MS mutants of the wildtype, E. coli DH5α.
Agent-based Fire diffuse fire model (ABFDF) and experimental validation of anomalous ion-channel mediated wave propagation in three-dimensional E. coli biofilm.
A) 3D spherical biofilm in a fluid-filled environment simulated using BSim. B) ABFDF global electrical signaling wavefront profile averaged over a three-dimensional biofilm. The ThT intensity is predicted as a function of time. C) Plot of the square radial distance of the wave front (R2) against time and fit with a power law, 
Fit constants for eqn 1 to results from the ABFDF and experimental data.
Nonlinear propagation of ion-channel mediated wave in 3D E. coli biofilms
A) Nonlinear relationship between propagation velocity of the wavefront and the time for (1) centrifugal wave and (2) centripetal wave of the first peak. B) Nonlinear relationship between propagation velocity of the wavefront and the radial distance for (1) centrifugal wave and (2) centripetal wave of the first peak.
