Bacterial cells that stop growing but maintain viability and the capability to regrow are termed dormant and have been shown to transiently tolerate high concentrations of antimicrobials. Links between tolerance and cellular energetics as a possible explanation for the tolerance, have been investigated and have produced mixed and seemingly contradictory results. Because dormancy merely indicates growth arrest, which can be induced by various stimuli, we hypothesize that dormant cells may exist in a range of energetic states that depend on the environment. To energetically characterize different dormancies, we first induce them in a way that results in dormant populations and subsequently measure both of their main energy sources, the proton motive force magnitude and the concentration of ATP. We find that different types of dormancy exhibit characteristic energetic profiles that vary in level and dynamics. The energetic makeup was associated with survival to some antibiotics but not others. Our findings portray dormancy as a state that is rich in phenotypes with various stress survival capabilities. Because environmental conditions outside of the lab often halt or limit microbial growth, a typologization of dormant states may yield relevant insights on the survival and evolutionary strategies of these organisms.

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments.

As an important free energy source, the membrane voltage (Vm) regulates many essential physiological processes in bacteria. However, in comparison with eukaryotic cells, knowledge of bacterial electrophysiology is very limited. Here, we developed a set of novel genetically encoded bacterial Vm sensors which allow single-cell recording of bacterial Vm dynamics in live cells with high temporal resolution. Using these new sensors, we reveal the electrically \"excitable\" and \"resting\" states of bacterial cells dependent on their metabolic status. In the electrically excitable state, frequent hyperpolarization spikes in bacterial Vm are observed, which are regulated by Na+/K+ ratio of the medium and facilitate increased antibiotic tolerance. In the electrically resting state, bacterial Vm displays significant cell-to-cell heterogeneity and is linked to the cell fate after antibiotic treatment. Our findings demonstrate the potential of our newly developed voltage sensors to reveal the underpinning connections between bacterial Vm and antibiotic tolerance.

The observation of neuron-like behavior in bacteria, such as the occurrence of electric spiking and extended bioelectric signaling, points to the role of membrane dynamics in prokaryotes. Electrophysiology of bacteria, however, has been overlooked for long time, due to the difficulties in monitoring bacterial bioelectric phenomena with those probing techniques that are commonly used for eukaryotes. Optical technologies can allow a paradigm shift in the field of electrophysiology of bacteria, as they would permit to elicit and monitor signaling rapidly, remotely, and with high spatiotemporal precision. In this perspective, we discuss about the potentiality of light interrogation methods in microbiology, encouraging the development of all-optical electrophysiology of bacteria.

Bacteria are electrically powered organisms; cells maintain an electrical potential across their plasma membrane as a source of free energy to drive essential processes. In recent years, however, bacterial membrane potential has been increasingly recognized as dynamic. Those dynamics have been implicated in diverse physiological functions and behaviors, including cell division and cell-to-cell signaling. In eukaryotic cells, such dynamics play major roles in coupling bioelectrical stimuli to changes in internal cell states. Neuroscientists and physiologists have established detailed molecular pathways that transduce eukaryotic membrane potential dynamics to physiological and gene expression responses. We are only just beginning to explore these intracellular responses to bioelectrical activity in bacteria. In this review, we summarize progress in this area, including evidence of gene expression responses to stimuli from electrodes and mechanically induced membrane potential spikes. We argue that the combination of provocative results, missing molecular detail, and emerging tools makes the investigation of bioelectrically induced long-term intracellular responses an important and rewarding effort in the future of microbiology.

Background: The use of electricity to mediate bacterial growth is unique in providing spatial control, but requires a more detailed understanding. Methods: We use two gold wires on a glass coverslip with an overlayer of agar to image Escherichia coli cells with brightfield and fluorescence microscopy while simultaneously applying a voltage. Cells outside of the wires provide a control population to measure cell growth as a function of voltage, rather than any difference in culture conditions or growth phase. Results: An applied voltage suppresses the fraction of E. coli undergoing elongation and division with recovery to control values when the voltage is removed. Depolarization is observed over the same voltage range suggesting a membrane potential-mediated response. Conclusions: Our experiments identify and use subcytotoxic voltages to measure differences in the fraction of E. coli cells elongating and dividing as a function of applied voltage. It is hoped that this research will inform the developing field of bacterial electrophysiology.

The bacterial flagellar motor (BFM) is a rotary molecular motor embedded in the cell membrane of numerous bacteria. It turns a flagellum which acts as a propeller, enabling bacterial motility and chemotaxis. The BFM is rotated by stator units, inner membrane protein complexes that stochastically associate to and dissociate from individual motors at a rate which depends on the mechanical and electrochemical environment. Stator units consume the ion motive force (IMF), the electrochemical gradient across the inner membrane that results from cellular respiration, converting the electrochemical energy of translocated ions into mechanical energy, imparted to the rotor. Here, we review some of the main results that form the base of our current understanding of the relationship between the IMF and the functioning of the flagellar motor. We examine a series of studies that establish a linear proportionality between IMF and motor speed, and we discuss more recent evidence that the stator units sense the IMF, altering their rates of dynamic assembly. This, in turn, raises the question of to what degree the classical dependence of motor speed on IMF is due to stator dynamics vs. the rate of ion flow through the stators. Finally, while long assumed to be static and homogeneous, there is mounting evidence that the IMF is dynamic, and that its fluctuations control important phenomena such as cell-to-cell signaling and mechanotransduction. Within the growing toolbox of single cell bacterial electrophysiology, one of the best tools to probe IMF fluctuations may, ironically, be the motor that consumes it. Perfecting our incomplete understanding of how the BFM employs the energy of ion flow will help decipher the dynamical behavior of the bacterial IMF.

Detecting live bacteria is an important task for antimicrobial susceptibility testing (AST) in the medical sector and for quality-monitoring in biological industries. Current methods for live-bacteria detection suffer limitations in speed or sensitivity. In a recent paper, we reported that electrical response dynamics in membrane potential enable single-cell rapid detection of live bacteria. The electrical response can be observed within a minute after electrical stimulation. Thus, it has potential in accelerating AST and the monitoring of biological samples. This method also enables experiments for biophysical and microbiological investigations into bacterial electrophysiology. With the hope that more researchers, scientists and engineers will use electrical stimulation for their assays, here we detail each step of the electrical stimulation experiment.

Extracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such as Shewanella oneidensis MR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET by S. oneidensis MR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell-level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with EET. Increasing electrode potential and associated EET current leads to more negative membrane potential. This EET-induced membrane hyperpolarization is spatially limited to cells in contact with the electrode and within a near-electrode zone (<30 μm) where the hyperpolarization decays with increasing cell-electrode distance. The high spatial and temporal resolution of the reported technique can be used to study the single-cell-level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

All cellular membranes have the functionality of generating and maintaining the gradients of electrical and electrochemical potentials. Such potentials were generally thought to be an essential but homeostatic contributor to complex bacterial behaviors. Recent studies have revised this view, and we now know that bacterial membrane potential is dynamic and plays signaling roles in cell-cell interaction, adaptation to antibiotics, and sensation of cellular conditions and environments. These discoveries argue that bacterial membrane potential dynamics deserve more attention. Here, we review the recent studies revealing the signaling roles of bacterial membrane potential dynamics. We also introduce basic biophysical theories of the membrane potential to the microbiology community and discuss the needs to revise these theories for applications in bacterial electrophysiology.