Enhancement of survival and electricity production in an engineered bacterium by light-driven proton pumping

Abstract

Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane potential and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependent proton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probe microbial physiology and to engineer microbes with new light-driven properties. Here, we describe functional expression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanella oneidensis strain MR-1. We report that there were significant increases in electrical current generation during illumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidence that an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistent with the hypothesis that proteorhodopsin activity enhances lactate uptake by increasing the proton motive force. Our results demonstrate that there is coupling of a light-driven process to electricity generation in a nonphotosynthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium under nutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain the widespread distribution of proteorhodopsin in marine environments.

FIG. 1.

Expression of functional PR by S. oneidensis. (A) Cell pellets of S. oneidensis expressing PR (+) or containing an empty control plasmid (−) grown overnight at 30°C in LB medium supplemented (+) or not supplemented (−) with retinal. Cells expressing PR in the presence of retinal have additional red pigment. (B) Difference in the absorption spectra of cultures of S. oneidensis expressing PR and cultures of S. oneidensis containing an empty control plasmid grown anaerobically in minimal medium containing 10 μM retinal.

FIG. 2.

Illumination of S. oneidensis expressing PR increases the membrane potential: histograms of red fluorescence intensities measured using flow cytometry for aerobic cultures of S. oneidensis expressing PR stained with the cyanine dye DioC₂(3). Cultures grown to an OD₆₀₀ of 1.0 in minimal medium with lactate were washed and then were resuspended in minimal medium without lactate. After 40 h, the cells starved of an electron donor were diluted into medium containing 0 or 10 mM lactate, were incubated for 1 h either in the dark or in the light, and then were stained and analyzed. Starved cultures incubated in the dark without lactate (green trace) contained both energized and deenergized cells; cultures incubated in the dark with lactate (blue trace) and cultures incubated in the light without lactate (red trace) contained only energized cells; addition of CCCP to the cells incubated in the light depolarized the membrane potential (black trace).

FIG. 3.

PR extends the viability of S. oneidensis during stationary growth. Cell growth was monitored by measuring the optical density at 600 nm for anaerobic cultures of S. oneidensis expressing PR in the presence of 3 mW cm⁻² of light (squares) or in the dark (circles). Cultures of S. oneidensis were incubated anaerobically at 30°C in minimal medium containing lactate, fumarate, and retinal. The values are means ± standard deviations for three independent cultures.

FIG. 4.

Light-dependent current increases in electrochemical chambers containing S. oneidensis expressing PR. (A) LED arrays used for illumination and glass cone of the electrochemical chamber containing both the working electrode and the counter electrode. So that details of the interior of the chamber could be seen, it was necessary to reduce the light intensity compared to the intensity used during the experiments and to remove reflections of the LEDs on the glass surfaces. (B) Oxidation current of an electrochemical chamber inoculated with S. oneidensis expressing PR (red trace) or containing a control plasmid (black trace). The arrows indicate the beginning of each 1-h illumination period. The light intensity was 10 mW cm⁻², and the traces are representative of three independent experiments.

FIG. 5.

Magnitude of the oxidation current depends on the light intensity. The change in oxidation current was measured for an electrochemical chamber containing an electrode fully colonized (>70 h) by cells of S. oneidensis expressing PR during illumination using the following light intensities: 0.7 mW cm⁻² (black trace), 1.9 mW cm⁻² (blue trace), 3.6 mW cm⁻² (green trace), and 9.0 mW cm⁻² (red trace). Each illumination period was 1 h long, and the current was allowed to stabilize before the next experiment. The arrows indicate the beginning and end of the illumination period.

FIG. 6.

Current for an electrochemical chamber containing S. oneidensis expressing PR during periods of constant darkness or constant light. After inoculation of the electrochemical chamber and attachment to the electrode, planktonic cells and the medium surrounding the electrode were removed and replaced with fresh medium (zero time). After a second medium exchange (at 67 h), the chamber was illuminated continuously using a light intensity of 10 mW cm⁻².

FIG. 7.

Schematic diagram of current generation by S. oneidensis expressing PR under anaerobic conditions with lactate as a carbon source. Excitation of PR by light drives the transport of protons (H⁺) across the membrane to increase the proton membrane potential, which is coupled to the import of lactate and synthesis of ATP. Oxidation of lactate leads to production of acetate, ATP, and reduced electron carriers, such as NADH and menaquinol. The enzymes that likely are involved in reducing the electron carriers include lactate dehydrogenase, NADH dehydrogenase, and formate dehydrogenase; the reactions may also transport protons. Electrons are passed through the Mtr extracellular respiratory pathway to the electrode (37).