Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and intercellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cellular proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane-potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namely Bacillus subtilis and Escherichia coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5-second electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K+ and the deletion of the K+ channel suggested that the hyperpolarization response is caused by the K+ efflux through the channel. When cells are preexposed to 400 ± 8 nm wavelength light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh–Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore, or alcohol. Therefore, electrically induced membrane-potential dynamics offer a reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level.

11Membrane-potential dynamics mediate bacterial electrical signaling at both intra-and inter-cellular 12 levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular 13 response to external electrical stimuli is influenced by the cell's proliferative capacity. A new strategy 14 enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane 15 potential dynamics would allow bridging this knowledge gap and further extend electrophysiological 16 studies into the field of microbiology. Here we report that an identical electrical stimulus can cause 17 opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated 18using two model organisms, namely B. subtilis and E. coli, and by developing an apparatus enabling 19exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we 20show that a 2.5 sec electrical stimulation causes hyperpolarization in unperturbed cells. Measurements 21 of intracellular K + and the deletion of the K + channel suggested that the hyperpolarization response is 22caused by the K + efflux through the channel. When cells are pre-exposed to UV-violet light, the same 23 electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model 24 extended from the FitzHugh-Nagumo neuron model suggested that the opposite response dynamics are 25 due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only 26induced depolarization when cells are treated with antibiotics, protonophore or alcohol. Therefore, 27electrically induced membrane potential dynamics offer a novel and reliable approach for rapid detection 28 of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell 29 level. 30 31

Engineering spatially organized biofilms for creating adaptive and sustainable biomaterials is a forthcoming mission of synthetic biology. Existing technologies of patterning biofilm materials suffer limitations associated with the high technical barrier and the requirements of special equipment. Here we present controlled meniscus-driven fluidics, MeniFluidics; an easily implementable technique for patterning living bacterial populations. We demonstrate multiscale patterning of living-colony and biofilm formation with submillimetre resolution. Relying on fast bacterial spreading in liquid channels, MeniFluidics allows controlled anisotropic bacterial colonies expansion both in space and time. The technique has also been applied for studying collective phenomena in confined bacterial swarming and organizing different fluorescently labelled Bacillus subtilis strains into a converged pattern. We believe that the robustness and low technical barrier of MeniFluidics offer a tool for developing living functional materials, bioengineering and bio-art, and adding to fundamental research of microbial interactions.

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