Light-powering Escherichia coli with proteorhodopsin
Jessica M. Walter*†, Derek Greenfield*‡, Carlos Bustamante*†‡§¶?, and Jan Liphardt*†‡**
Departments of *Physics,§Chemistry, and¶Molecular and Cell Biology,?Howard Hughes Medical Institute, and‡Biophysics Graduate Group, University
of California, Berkeley, CA 94720; and†Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
Contributed by Carlos Bustamante, December 13, 2006 (sent for review December 1, 2006)
community sequencing of ocean samples. Previous studies have
but its role in powering cells and participation in ocean energy
fluxes remains unclear. Here, we show that when cellular respira-
tion 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 absorp-
tion 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 (103photons 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
challenges by harvesting light energy.
light-driven proton pumps ? oceanic bacteria
photosynthesis (1, 2). Other light-harvesting mechanisms include
the light-driven proton pump bacteriorhodopsin, used by halobac-
teria living in salt ponds to supplement respiration (3). In 2000, a
novel light-driven proton pump, proteorhodopsin (PR), was dis-
covered (4). The world’s oceans contain an estimated 1028PR-
expressing bacteria, placing them among the most prevalent organ-
isms on Earth (5, 6). Proteorhodopsins are widely distributed and
spectrally tuned to their oceanic environments (5, 7–12). PR is
distinguished from bacteriorhodopsin by its high sequence homol-
ogy to sensory rhodopsins (postulated to have evolved from a
different origin than bacteriorhodopsin; ref. 4), its presence in
members of Eubacteria (as opposed to Archaea), and significant
out of the cell (4).
Abundant evidence exists for PR’s function as a transmem-
brane proton pump, including light-mediated transport of pro-
of protons by illuminated PR?Escherichia coli cells (4, 7, 13).
Despite the ability of PR to pump protons, green light illumi-
nation did not increase SAR11 growth rates or cell yield in cell
culture experiments (14), leaving proteorhodopsin’s in vivo
contribution unclear. Moreover, the extent to which light-
harvesting by PR can complement or replace other cellular
energy sources remains to be quantified.
of protons across the membrane, maintained under aerobic
conditions by oxidative phosphorylation. Bacteria use the pmf to
synthesize ATP, drive chemiosmotic reactions, and power the
rotary flagellar motor (15, 16) In 1974, Racker and Stoeckenius
(17) demonstrated that the pmf generated by light-driven proton
pumping by bacteriorhodopsin could be used to produce ATP.
We reasoned that measuring the pmf of illuminated PR?cells
could identify a role of PR in cellular energy fluxes. The speed
quatic ecosystems play a major role in the conversion of light
energy into chemical energy, principally via chlorophyll-based
of the E. coli flagellar motor is proportional to the pmf over a
wide range of speeds (18, 19), therefore, proton extrusion by PR
should increase the flagellar motor’s rotation rate when the cells
are illuminated. We used E. coli as the heterologous host in our
PR experiments because it is the primary model organism for
studying Gram-negative bacteria and techniques for pmf mea-
surement and PR expression in E. coli have been refined (4, 5,
7–11, 13, 18, 19).
Results and Discussion
We tracked swimming PR?E. coli in two dimensions by using
dark-field microscopy, periodically illuminating the cells with
green light at PR’s absorption maximum. We observed single
cells to characterize rapid responses of the cellular pmf to light.
No detectable increase in cell swimming velocity occurred upon
illumination with green light. We surmised that light-driven
proton pumping may benefit the cell only under certain envi-
ronmental conditions, as suggested by Giovannoni et al. (14).
To test the possibility that light-driven proton pumping is
most beneficial to aerobically grown cells when their ability to
respire is suddenly impaired, we energy-depleted the cells.
Because E. coli is difficult to energy-deplete by nutrient
limitation because of its endogenous energy stores (20, 21), we
additionally used the respiratory poison azide, which has
multiple cellular effects (22, 23) but primarily inhibits cyto-
chrome oxidase and, thus, proton extrusion by the respiratory
chain, stopping the flagellar motor (18).
Strikingly, with respiration inhibited by azide, PR?cells
responded to green light. PR?cells in 30 mM azide swam slowly
in red illumination, but they showed a marked velocity increase
with green illumination (Fig. 1b). Upon removal of the green
light, they slowed to their previous velocity. To increase the
accuracy of our flagellar rotation measurements, we subse-
quently used a tethered cell geometry (Fig. 2a), permitting
extended observation of the same bacterium in different illumi-
nation conditions. PR?cells were allowed to stick to the
coverslip via a flagellum, and we then monitored the angular
rotation rate of cells (Fig. 2a). A typical tethered cell rotated at
a mean rate of 0.2–1 Hz, depending on its length and the position
of the stuck flagellum along its body. To facilitate data inter-
pretation, we deleted the cheY gene (24, 25), yielding smooth-
swimming mutants whose flagellar motors do not reverse.
As expected, there was no effect of green light on the cell’s
rotation rate in the absence of azide (Fig. 2b). However, as we
inhibited respiration by adding azide, the cells again became
light-responsive. PR?cells sped up upon illumination with green
light [Fig. 2 a and c, see also supporting information (SI) Movie
1]. The PR?cells were converting light energy into an electro-
Author contributions: J.M.W. designed research; J.M.W. performed research; J.M.W., D.G.,
and J.L. wrote the paper; D.G. analyzed data; D.G. performed model simulations; and C.B.
and J.L. conceived the experiment.
The authors declare no conflict of interest.
Abbreviations: pmf, proton motive force; PR, proteorhodopsin.
**To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
February 13, 2007 ?
vol. 104 ?