Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage-Indicating Protein

Abstract

Bacteria have many voltage- and ligand-gated ion channels, and population-level measurements indicate that membrane potential is important for bacterial survival. However, it has not been possible to probe voltage dynamics in an intact bacterium. Here we developed a method to reveal electrical spiking in Escherichia coli. To probe bacterial membrane potential, we engineered a voltage-sensitive fluorescent protein based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) in E. coli revealed electrical spiking at up to 1 hertz. Spiking was sensitive to chemical and physical perturbations and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated.

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

“…Using the assay of growth (rate), we were then able to demonstrate an improvement in growth of the transgenic Synechocystis strain expressing PR, when compared with the control strain expressing a non-pumping proteorhodopsin, PROPS (35). To our knowledge, this is the first demonstration of a beneficial effect of PR on the growth of a cyanobacterium.…”

Directed evolution of a far-red fluorescent rhodopsin

“…Bacterial phytochrome receptors have recently emerged as potential substrates for the development of fluorescent markers in the NIR range (4,13,25). These proteins, which use biliverdin (an intermediate of heme metabolism) as a chromophore, can be engineered to excite and emit well into the infrared (termed iRFPs) (26).…”

“…In the field of optogenetics, rhodopsins have been used as actuators of neuronal activity: When certain rhodopsin variants are expressed in neurons, light-driven ion flux can be exploited to activate (9) or inhibit (10) neuronal activity with fast kinetics in a selective and reversible fashion. The absorbance spectrum of rhodopsins can be sensitive to membrane voltage (11), and at least three rhodopsins have been reported to exhibit dim fluorescence that is sensitive to changes in membrane voltage (3,4,12), a property that has been harnessed to develop genetically encoded fluorescent sensors of neuronal activity (3,12,13). Although significant progress has been made in developing opsin-based tools that enable selective activation/inhibition of neurons, progress toward developing brighter opsin-based biological sensors has been limited.…”

“…Microbial opsins are integral membrane proteins with seven transmembrane helices and a conserved lysine residue that forms a covalent Schiff-base linkage to all-trans retinal. The apoprotein covalently bonded to a retinal molecule forms a colored holoprotein referred to as rhodopsin (2), variants of which have recently been reported to naturally fluoresce at wavelengths >680 nm in live cells (3,4). Rhodopsins thus offer novel substrates for engineering genetically encoded fluorescent markers in a desirable spectral range for livecell imaging.…”

Emotion: The self-regulatory sense

“…Indeed, voltage spiking has recently been observed in the E coli, with on/off "blinking" associated with aerobic respiration and the stress response. 229 Likewise the dynamic growth and shrinkage of the spines themselves follows the same pattern of regulatory increases and decreases (of specialized glutamate receptors) associated with long-term potentiation and damping, correlating with synaptic plasticity, the "self-modifying" cognitive processes that give rise to memory, emotion and executive function 230 -core elements of human consciousness. Indeed, spine plasticity itself responds to life experience including fear conditioning 231 , and intriguingly -as with the aforementioned epigenetic methylation marksaltered or disordered spine dynamics, morphology or density, are associated with psychiatric diseases and neurological degeneration.…”