Electrical and Optical Control of Bacterial Membrane Potential and Growth

Bacteria maintain a resting membrane potential which is generated from the ion gradients across the membrane. Membrane potential is important for bacterial functions, such as ATP synthesis, cell transport, cell proliferation and division, cell-cell communication, and antibiotic resistance. Controlling bacterial membrane potential and cell growth has many potential applications for anti-bacterial agents, synthetic biology and living materials. A range of solution-phase agents, such as antibiotics and ionophores, can be used to finely tune the bacterial membrane potential and growth, but these agents all lack spatial control. Developing methods with better spatial control of bacterial membrane potential and cell growth is the original goal of our research. We also try to understand the mechanisms of our control methods and elucidate the relationship between bacterial membrane potential and cell growth.Electric fields have been widely used in neuroscience for modulating neuron activities and treating brain disorders such as Parkinson's disease. They have better spatial precision than solution-phase agents. Previous research mainly used electric fields for electroporation and antibacterial applications. However, the relationship between applied electric field and bacteria growth has not been well characterized. We developed a device which allows the application of an electric field while imaging cell growth and membrane potential change simultaneously. We discovered a range of low frequency voltages that induce slower bacteria growth with increased voltage without killing the cells; subsequently, the bacteria can recover to normal growth levels after removal of the voltage. Hyperpolarization waves can be visually observed during the application of an electric field to bacteria. We identified that gold ions, electrochemically-generated from the gold electrodes in our experiments, are the main cause of the observed slow bacterial growth and hyperpolarization. The speed of the hyperpolarization wave can be modulated by adjusting the applied voltage and frequency, which controls the rate of gold ions electrochemically-generated from electrodes, confirmed by inductively coupled plasma mass spectrometry. Solution-phase gold ion salts were shown to similarly slow bacteria growth and induce hyperpolarization, further validating our observations. To eliminate the effect of side reactions and have better spatial control, we moved to using blue light. Blue light exposure has been demonstrated to hyperpolarize bacteria and encode membrane potential based patterns within a biofilm. Additionally, high doses of blue light can inactivate microbes. Despite this work, using blue light to control bacteria growth at a single cell level has not been previously studied. We have discovered that in the sub-cytotoxic range (30-50 s, 480 nm), longer blue light exposure leads to slower bacterial growth without inducing measurable cell death. Exposure areas can be tightly controlled by moving the light beam of a fluorescence microscope. As a result, complex patterns can be achieved in growing bacterial communities by locally limiting bacteria growth. Our results suggest that the mechanism of blue light control on bacteria growth may be related to hyperpolarization, generation of reactive oxygen species, and increased esterase activities, but future works are needed to decouple these different factors. Notably, we also found that the commonly used Nernstian dye Thioflavin T (ThT) slows the growth of bacteria and may lead to previously-unconsidered experimental artifacts. Cells hyperpolarized by blue light (3 s, 480 nm) internalize more ThT, leading to higher fluorescence signals in these cells than unexposed controls. Additionally, hyperpolarized cells grow slower than control cells in the presence of ThT; however, in the absence of ThT, blue light (3 s, 480 nm) exposed cells do not have much difference of growth compared to unexposed cells. These results suggest that difference of intracellular ThT concentration rather than hyperpolarization is the main reason of slowed bacteria growth when ThT is used as the membrane potential indicator. In summary, we discovered that solution-phase and electrochemically-generated gold ions lead to the hyperpolarization of bacteria and slow cell growth, it provides a new tool for controlling bacterial electrophysiology. This finding may also relate to the antibacterial study of gold nanoparticles. In addition, we figured out that the widely used Nernstian dye ThT slows bacterial growth and causes previously unconsidered experimental artifacts in bacteria growth study. Last, we found that blue light causes slow growth of bacteria and can be used to pattern engineered living materials. Future works are needed to understand the mechanism of the slow bacterial growth induced by blue light.