Harvard researchers have engineered a genetically encoded voltage-indicating protein to measure the membrane potential (Vm) of bacteria cells, introducing a tool to explore the relatively new field of bacterial electrophysiology.
By reversing the function the light-driven protein pump called green-light absorbing proteorhodopsin (GPR), Harvard associate professor Adam Cohen and his team engineered a proteorhodopsin optical proton sensor (PROPS) that lights up in response to electrical activity within Escherichia coli cells.
The optical signal from PROPS however is very dim, about 1000 times dimmer than GFP and undetectable with conventional epifluorescence microscopes. Therefore the group adapted a single-molecule fluorescence microscopy system to detect the signal. The system consisted of an inverted microscope, an intense laser designed for wide-field illumination, a high NA objective, a good filter set, and a very sensitive camera.
When viewed under their adapted microscope, Cohen and colleagues saw the engineered E. coli cells blinking, in apparently random patterns of intensity and periodicity.
“The mysterious sudden spontaneous drops in such a fundamental bioenergetic parameter indicate that there is a very important aspect of bacterial physiology that has not previously been recognized,” says John Spudich, director of The Center for Membrane Biology at the University of Texas Medical School who studies photosensory receptors.
Before introducing their voltage-indicating protein, Cohen and his group needed to confirm that the PROPS was actually detecting the cells’ Vm. “We had an unknown sensor, and we were looking at an unknown biological phenomenon,” said Cohen. In an experiment designed to indirectly calibrate PROPS, individual bacteria cells were tethered to a coverslip by their flagellum. Torquing cells, powered by the protonmotive force, were observed to slow their rotation in correlation to changing brightness.
Because of the extremely small scale and the presence of the cell wall, traditional methods of electrophysiology such as patch clamping are not applicable to bacteria. Attempts to measure bacterial Vm via patch clamping requires degrading the cell wall and ballooning the cells into spheroplasts, steps that disrupt the cell’s functions.
The study suggests that the probe could be used to study the electrical regulation of efflux machinery, a common pathway of microbial resistance. “Blinking was correlated with efflux of a positively charged dye molecule,” Cohen said. “Every time the cells blinked, the level of the dye would drop precipitously.”
Now, Cohen’s group plans to screen knockout E. coli strains for altered blinking behaviors to determine the functions of various proteins and their role in Vm. “When we measure voltage, we’re measuring the aggregate effect of large number of proteins,” said Cohen.
The paper, “Spiking in Escherichia coli probed with a fluorescent voltage-indicating protein,” was published in the 15 July 2011 issue of Science.