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Link to original content: https://pubmed.ncbi.nlm.nih.gov/17277079/
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. 2007 Feb 13;104(7):2408-12.
doi: 10.1073/pnas.0611035104. Epub 2007 Feb 2.

Light-powering Escherichia coli with proteorhodopsin

Jessica M Walter  1 , Derek Greenfield, Carlos Bustamante, Jan Liphardt
Affiliations

Light-powering Escherichia coli with proteorhodopsin

Jessica M Walter et al. Proc Natl Acad Sci U S A. .

Abstract

Proteorhodopsin (PR) is a light-powered proton pump identified by community sequencing of ocean samples. Previous studies have established the ecological distribution and enzymatic activity of PR, but its role in powering cells and participation in ocean energy fluxes remains unclear. Here, we show that when cellular respiration is inhibited by depleting oxygen or by the respiratory poison azide, Escherichia coli cells expressing PR become light-powered. Illumination of these cells with light coinciding with PR's absorption spectrum creates a proton motive force (pmf) that turns the flagellar motor, yielding cells that swim when illuminated with green light. By measuring the pmf of individual illuminated cells, we quantify the coupling between light-driven and respiratory proton currents, estimate the Michaelis-Menten constant (Km) of PR (10(3) photons per second/nm2), and show that light-driven pumping by PR can fully replace respiration as a cellular energy source in some environmental conditions. Moreover, sunlight-illuminated PR+ cells are less sensitive to azide than PR- cells, consistent with PR+ cells possessing an alternative means of maintaining cellular pmf and, thus, viability. Proteorhodopsin allows Escherichia coli cells to withstand environmental respiration challenges by harvesting light energy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Light-powering of PR+ E. coli. (a) Overview of spectra and spectral overlaps. The PR absorption spectrum [redrawn from Beja (4)] is shown in black, with green and red bars indicating illumination wavelengths. (b) Single PR+ bacterium swims faster when illuminated with green light. The cell's position is recorded at a rate of 0.5 Hz via constant red dark-field illumination throughout the entire track. Illumination with green light at the absorption peak is periodic (20 s on, 20 s off) and occurs only during green circles of the path. Velocity during periods of green illumination (10.6 ± 0.9 μm/s) is 96% higher than during periods of red illumination alone (5.4 ± 0.4 μm/s; P < 0.005, one-tailed t test). Respiration has been inhibited with 30 mM azide in motility buffer. (Inset) Raw tiled movie frames of swimming cell.
Fig. 2.
Fig. 2.
Flagellar response to green light measured in PR+ and PR− bacteria observed at different levels of respiratory inhibition. (a) Movie frames showing a tethered cell in red light and green light (full movie is available at SI Movie 1), a plot of the angular position versus time of a cell in 60 mM azide, and a schematic of tethered cell geometry. In red light, the cell rotationally diffuses about its attachment point. Green light (shaded area) leads to counter clockwise rotation of the cell. (b and c) Cells without (b) and with (c) PR. Solid, dotted, and dashed lines show three representative cells per condition. Note the considerable cell-to-cell variation in absolute rotation rates due to variation of cell length and tethering geometry. With or without azide, cells lacking PR show no response to green light. Removal of oxygen also leads to light-responsiveness. At high azide concentrations (c Top), angular velocity is nearly zero until cells are illuminated with green light.
Fig. 3.
Fig. 3.
The response of PR+ bacteria to green light increases with respiratory inhibition and light intensity. (a) Benefit of illumination versus degree of inhibition of the respiratory system. The difference between angular velocity in green light and red light (ωG–ωR) becomes pronounced in PR+ bacteria (filled circles) as respiration is inhibited by low oxygen or sodium azide. PR− cells (open circles) show no change between red and green illumination. To facilitate comparison between cells, the angular velocities are normalized by each bacterium's maximum velocity. n = 5–14 cells per condition. Green line, fit to model described in d. (b) The rotation speed of PR+ cells depends on the intensity of green illumination. Individual PR+ spinner cells were exposed to six intensities of green light. The mean angular velocity at each intensity is plotted (n = 5–6 cells for each intensity), normalized by the velocity at maximum illumination. Dashed lines, fits to model described in d. (c) Overview of transmembrane fluxes and proton pumping in PR+ E. coli. Sources of proton motive force include respiration and PR. Sinks include rotation of the flagellar motor and ATP synthesis. (d) Model including sources of pmf (respiration and proteorhodopsin), sinks (such as the flagellar motor), and the membrane capacitance. The variable resistors RR and RPR model the effect of azide and light on proton extrusion by respiration and PR, respectively. The voltmeter (top-most circuit element) measures the potential difference across the membrane (equivalent to the pmf).
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