APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2010, p. 4123–4129 Vol. 76, No. 130099-2240/10/$12.00 doi:10.1128/AEM.02425-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Enhancement of Survival and Electricity Production in an EngineeredBacterium by Light-Driven Proton Pumping�†

Ethan T. Johnson,1 Daniel B. Baron,2 Belén Naranjo,2 Daniel R. Bond,2,3Claudia Schmidt-Dannert,1* and Jeffrey A. Gralnick2,3*

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 140 Gortner Laboratory,1479 Gortner Avenue, St. Paul, Minnesota 551081; BioTechnology Institute, University of Minnesota, 140 Gortner Laboratory,

1479 Gortner Avenue, St. Paul, Minnesota 551082; and Department of Microbiology, University of Minnesota,MMC 196, 420 Delaware Street SE, Minneapolis, Minnesota 554553

Received 6 October 2009/Accepted 20 April 2010

Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane poten-tial and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependentproton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probemicrobial physiology and to engineer microbes with new light-driven properties. Here, we describe functionalexpression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanellaoneidensis strain MR-1. We report that there were significant increases in electrical current generation duringillumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidencethat an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistentwith 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 nonphoto-synthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium undernutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain thewidespread distribution of proteorhodopsin in marine environments.

Classic experiments in microbial bioenergetics used light-driven reactions from halobacterial bacteriorhodopsin or thephotosynthetic reaction center to provide a temporary drivingforce for understanding transport and chemiosmotic coupling(6, 7, 19, 35). However, light-driven reactions have not beenused in metabolic engineering to alter microbial physiologyand production of chemicals. The recent discovery of proteo-rhodopsin (PR) in ocean microorganisms and the ease withwhich this membrane protein can be functionally expressed byrecombinant bacteria have made possible many engineeringstrategies previously not available (1, 16). In this paper, wedescribe progress toward the goal of integrating light-drivenreactions with biocatalysis.

In contrast to the situation for established industrial micro-organisms, such as Escherichia coli, our current understandingof less-studied algal and phototrophic bacteria may limit met-abolic engineering strategies which require genetic manipula-tion. Metabolic engineering strategies using photosyntheticbacteria have focused largely on methods to increase hydrogenproduction, and improvements rely mainly on engineering ofnitrogenase and hydrogenase to produce H2. Algae appear to

be suited to large-scale cultivation for lipid production, but sofar little has been done to engineer these organisms (36). Inprinciple, platform microbial hosts capable of producing a di-verse range of products could be boosted by addition of light-driven processes from phototrophic metabolism.

To demonstrate the feasibility of transferring a light-drivenprocess into a nonphotosynthetic bacterium, we chose to studyproteorhodopsin (PR) first because it is one of the simplest mech-anisms for harnessing the energy from light. The proteorho-dopsins are a group of transmembrane proteins that use thelight-induced isomerization of retinal, the oxidative cleavageproduct of the carotenoid �-carotene, either to initiate signalingpathways or to catalyze the transfer of ions across cell membranes(8). PR was discovered by metagenomic analysis of marine sam-ples (1) and is related to the well-studied bacteriorhodopsin ofarchaea (33) and rhodopsin (34), a eukaryotic light-sensing pro-tein. The membrane potential generated by light-driven protonpumping by PR has been confirmed to drive ATP synthesis in aheterologous system (25). However, bacteria expressing heterol-ogous PR were shown not to benefit from this pumping activity,as no significant increases in growth rates were observed (9). Thisled to the suggestion that PR may benefit the organism onlyunder starvation conditions. In agreement with this hypothesis,Gomez-Consarnau et al. (10) have reported that the light-depen-dent growth rates of a marine flavobacterium that has a native PRare increased only when the organism is cultured under energy-limited conditions.

Studies of both native and recombinant systems in whichrhodopsins are expressed have generated light-dependentmembrane potentials. In membrane vesicles isolated from anative host, the light-dependent membrane potential gener-

* Corresponding author. Mailing address for Jeffrey A. Gralnick:BioTechnology Institute, University of Minnesota, 140 Gortner Labo-ratory, 1479 Gortner Avenue, St. Paul, MN 55108. Phone: (612) 626-6496. Fax: (612) 625-5780. E-mail: gralnick@umn.edu. Mailingaddress for Claudia Schmidt-Dannert: Department of Biochemistry,Molecular Biology and Biophysics, University of Minnesota, 1479Gortner Avenue, St. Paul, MN 55108. Phone: (612) 625-5782. Fax:(612) 625-5780. E-mail: schmi232@umn.edu.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 7 May 2010.

4123

ated by bacteriorhodopsin provides the driving force for ATPsynthesis (35) and uptake of leucine and glutamate (20, 22).More recently, studies of recombinant systems have coupledthe membrane potential to other transport processes. In oneexample, the membrane potential-dependent export of specifictoxic molecules increased when E. coli cells expressing both anarchaeal rhodopsin and a specific efflux pump were exposed tolight (17). In another experiment, starved E. coli cells express-ing PR increased the swimming motion of their flagella whenthey were illuminated (44). Based upon measurements offlagellar motion as a function of light intensity and azide con-centration, the proton motive force generated by PR was esti-mated to be �0.2 V, a value similar to the value for aerobicrespiration in E. coli (42).

As a nonphotosynthetic host for recombinant PR expression,we chose the dissimilatory metal-reducing bacterium Shewanellaoneidensis strain MR-1, which is genetically tractable for engi-neering and is able to use a variety of terminal electron acceptors,including insoluble metal oxides (11, 30). Key to the ability of thisbacterium to reduce metal oxides is a multicomponent extracel-lular respiratory pathway that transports electrons from menaqui-nol to cytochromes in the outer membrane. This pathway is com-posed of a cytoplasmic membrane tetraheme protein (CymA), aperiplasmic decaheme protein (MtrA), an integral outer mem-brane protein (MtrB), and a decaheme lipoprotein (MtrC) that isassociated with MtrB (14, 37, 40). The ability of S. oneidensis toreduce extracellular metal oxides has made it possible to harvestelectrons from this organism by coupling it to an electrode whichserves as the electron acceptor (21). The electron flow to theouter surface allows respiration rates to be measured directly byelectrochemistry.

In the current work, we introduced PR into an electricity-generating bacterium, S. oneidensis strain MR-1, and demon-strated that there was integration of a light-driven process intothe metabolism of a previously nonphotosynthetic organismthat resulted in a useful output. We show here that PR allowscells to survive for extended periods in stationary phase andthat the presence of light results in an increase in electricitygeneration. A possible physiological model to explain theseeffects is discussed.

MATERIALS AND METHODS

Microbiological methods. E. coli JM109 was used for all molecular biologyprocedures, and plasmids were transferred to S. oneidensis strain MR-1 byconjugation with the mating strain E. coli WM3064. S. oneidensis was cultivatedat 30°C either in Luria-Bertani (LB) medium or in minimal medium containing20 mM lactate (15, 23). Anaerobic cultures of S. oneidensis contained 40 mMfumarate as an electron acceptor when they were not grown in electrochemicalchambers with an electrode as the electron acceptor. When required, 50 �g ml�1

kanamycin and 10 �M retinal were included for plasmid selection and PRreconstitution, respectively. Optical densities at 600 nm (OD600) of cell cultureswere determined with a Bausch and Lomb Spectronic 20. Estimates of thenumber of cells ml�1 in a culture were obtained by counting the number ofcolonies after a dilution series of the culture was plated on LB agar.

Gene cloning. The PR gene of a marine bacterium in the SAR86 phylogeneticgroup (original contig GenBank accession no. AF279106; proteorhodopsin geneGenBank accession no. AAG10475) was amplified by PCR from a plasmidcontaining the PR gene (1) using gene-specific oligonucleotides (5�-GGTCTAGAAGGAGGAGATCTACATATGAAATTATTACTGATATTAG-3� and 5�-ACTAGCGGCCGCTTAAGCATTAGAAGATTCTTTAAC-3�) with addedXbaI and NotI restriction sites and a Shine-Dalgarno sequence. The PCR prod-uct was cloned into the constitutive expression vector pUCmod (the lac repressorbinding sequence was deleted) (39) and sequenced to ensure that the PR gene

was not changed. The PR gene and the upstream constitutive lac promoter thenwere subcloned into the BamHI site of the broad-host-range vectorpBBR1MCS-2 (18) for expression in S. oneidensis (3). Subsequent digestion ofthe pBBR1MCS-2 plasmid containing the fragment cloned into the BamHIrestriction site revealed that the modified promoter and PR gene were orientedopposite the endogenous pBBR1MCS-2 promoter sequence.

PR absorption spectroscopy. Cultures of S. oneidensis harboring pBBR1MCS-2 con-taining the PR gene or an empty control plasmid were incubated anaerobically inminimal medium containing 10 �M retinal at 30°C for approximately 16 h. Thecells were centrifuged and resuspended in 50 mM Tris-HCl (pH 8), 0.1 M NaCl.It was difficult to measure the difference spectrum because of the backgroundcaused by scattering and absorption by native cytochromes expressed by S.oneidensis. To reduce the scattering of a sample, resuspended cells were soni-cated using 10-s pulses at 30% power for a total of 90 s (Branson Sonifier).Absorption spectra were measured (Varian CARY 50 spectrophotometer), andthe effects of scattering were removed by subtracting a cubic polynomial fit to thedata at 310 nm and 700 to 750 nm. To remove absorption by native cytochromes,the difference spectrum for cultures of S. oneidensis containing pBBR1MCS-2with the PR gene and cultures of S. oneidensis containing an empty controlplasmid was calculated. To account for minor differences in cytochrome absorp-tion at 400 nm and the numbers of cells in each culture, the two baseline-corrected spectra were normalized at the cytochrome absorption peak (�max, 410nm) by scaling the spectrum for the culture of S. oneidensis containing the emptyplasmid. To estimate the number of PR molecules cell�1, the concentration ofPR in the sample was calculated by assuming that the molar extinction coefficientat 520 nm was 50,000 M�1 cm�1 (2), and the number of viable cells wasdetermined by plating serial dilutions on agar plates. The estimate was based onthree replicate experiments

Illumination methods. Light for both the growth rate measurement experi-ments and the electrochemical experiments was generated by circuit boardscontaining arrays of green (�max, 530 nm) high-power light-emitting diodes(LEDs) (Luxeon III Emitter; LXHL-PM09; Future Electronics) powered by adirect current power supply (Mastech model HY3030E). In the growth experi-ments, the samples were illuminated by LEDs spaced approximately 3 cm apart.In the electrochemical experiments, both faces of the electrode were illuminatedby separate arrays of LEDs (see Fig. 4A). To provide sufficient light intensity, thecircuit boards were coated with thin layers of clear epoxy (EPO-TEK 302-3 M;Epoxy Technology) and were submerged in the temperature-controlled waterbath �1 cm from the electrochemical chamber. Light intensities were measuredwith a light meter (AEMC Instruments model CA813), and photometric units(lux) were converted to radiometric units (mW cm�2) using the standard pho-topic response and power spectrum of the LEDs.

Membrane potential measurement. The membrane potential for S. oneidensiswas measured using a membrane potential-sensitive dye and flow cytometry (31).Cells grown aerobically in minimal medium with 20 mM lactate were centrifugedand then washed and resuspended in an equal volume of minimal mediumwithout lactate as the electron donor. After incubation at 30°C for 30 to 40 h inthe dark, the cells were diluted to obtain a concentration of 106 cells ml�1 byaddition of minimal medium containing either 0 or 10 mM lactate. Followingincubation for 1 h at 30°C either in the dark or in the presence of approximately3 mW cm�2 of light, the cells were exposed to the cyanine dye DioC2(3) at aconcentration of 10 �M for 5 min in the dark. Fluorescence intensities atwavelengths greater than 640 nm were measured for 10,000 cells using aFACSCalibur flow cytometer (Becton Dickinson). An EDTA treatment that isoften required to disrupt the cell membrane and allow the dye to enter the cellsof Gram-negative bacteria was not required for S. oneidensis. For depolarizationof the membrane potential, the protonophore carbonyl cyanide 3-chlorophenyl-hydrazone (CCCP) was added at a concentration of 5 �M.

Electrochemical studies. Electrochemical measurements for S. oneidensis weredetermined using 20-ml electrochemical chambers as described previously (23, 24).Ten milliliters of an anaerobic culture of S. oneidensis grown to an OD600 of 0.4 to0.5 at 30°C in minimal medium containing 30 mM lactate, 40 mM fumarate, 50 �gml�1 kanamycin, and 10 �M retinal was transferred to a sterile, oxygen-sparged,stirred electrochemical chamber. During medium exchanges, the medium surround-ing the electrode was replaced with fresh medium containing 30 mM lactate, 50 �gml�1 kanamycin, 10 �M retinal, and 0.5 �M riboflavin (23). During periods of light,both faces of a graphite working electrode (AXF-5Q; Poco Graphite, Inc.) wereilluminated. For experiments in which periodic illumination was used, the electro-chemical chambers (with 10 ml of medium) contained electrodes that were 2 cm by0.5 cm by 0.1 cm; for metabolite experiments in which continuous illumination wasused, the electrochemical chambers (with 15 ml of medium) contained electrodesthat were 2 cm by 2 cm by 0.1 cm and the chambers were flushed with nitrogen gas.Heat from the LED circuit was dissipated using a temperature-controlled circulating

4124 JOHNSON ET AL. APPL. ENVIRON. MICROBIOL.

water bath (NESLAB, RTE-5B), and the measured temperature did not vary bymore than 0.3°C. Radiant heating due to light was negligible because the LED lightsources do not emit infrared light. The potential at the working electrode wasmaintained at 0.245 V versus standard hydrogen electrode, and the current produc-tion at 30°C was measured at 30-s intervals.

To measure metabolite concentrations in the medium from electrochemcialchambers, samples were removed from the chambers, centrifuged to removecells, and loaded (2 �l) onto an Agilent Zorbax SB-Aq C18 column (5 �m; 4.6 by250 mm), and chromatography was performed using an Agilent 1100 high-performance liquid chromatography (HPLC) system equipped with a photo-diode array detector. Metabolites were separated with an isocratic mobile phasecontaining 20 mM sodium phosphate (pH 2) and 0.5% acetonitrile at a rate of 1ml min�1, and the absorbance at 210 nm was monitored. Standard curves weregenerated using known concentrations of each chemical, and peak areas wereintegrated using the Agilent ChemStation software.

RESULTS

Functional expression of PR by S. oneidensis. We first estab-lished that expression and reconstitution of PR bound to retinal inS. oneidensis lead to accumulation of red pigment in the cellmembrane (Fig. 1A). While wild-type cultures are naturally pinkdue to expression of c-type cytochromes under O2-limiting con-ditions, quantitative changes in absorption due to functional re-constitution of PR were measured by calculating the differencespectrum for retinal-expressing PR cultures and cultures contain-ing an empty control plasmid (Fig. 1B). A comparison of thedifference spectrum obtained to previous PR spectra suggestedthat there was functional reconstitution of recombinant PR in S.oneidensis (1). Using an extinction coefficient for rhodopsins ab-sorbing in the visible region and the number of cells ml�1 culture,we estimated that there were 40,000 molecules of PR per S.oneidensis cell grown anaerobically in minimal medium. Our es-timate is similar to previous results reported for native PR ex-

pression in bacteria belonging to the SAR86 phylogenetic groupand in Pelagibacter cells, which contain 24,000 and 10,000 mole-cules of PR per cell, respectively (2, 9).

Having established that there was expression in S. oneidenis,we next examined generation of light-dependent membranepotentials by monitoring the fluorescence of cells labeled withthe lipophilic cyanine dye DioC2(3), which aggregates at highconcentrations, causing its fluorescence to shift to longer wave-lengths (31). Because the interior of energized cells has anegative potential relative to the exterior, the positivelycharged dye accumulates in the cytoplasm and the fluorescenceintensity at longer wavelengths increases. Membrane poten-tials of cells deenergized through starvation or addition of theprotonophore carbonyl cyanide 3-chlorophenylhydrazone(CCCP) become depolarized and no longer drive accumula-tion of the dye in the cytoplasm.

Cultures of S. oneidensis expressing PR were incubated in thedark without an electron donor to starve the cells and to deen-ergize the membrane potential. Cells then were incubated in thelight or in the dark with and without lactate as an electron donorand exposed to DioC2(3), and the fluorescence intensities of in-

FIG. 2. Illumination of S. oneidensis expressing PR increases themembrane potential: histograms of red fluorescence intensities mea-sured using flow cytometry for aerobic cultures of S. oneidensis ex-pressing PR stained with the cyanine dye DioC2(3). Cultures grown toan OD600 of 1.0 in minimal medium with lactate were washed and thenwere resuspended in minimal medium without lactate. After 40 h, thecells starved of an electron donor were diluted into medium containing0 or 10 mM lactate, were incubated for 1 h either in the dark or in thelight, and then were stained and analyzed. Starved cultures incubatedin the dark without lactate (green trace) contained both energized anddeenergized cells; cultures incubated in the dark with lactate (bluetrace) and cultures incubated in the light without lactate (red trace)contained only energized cells; addition of CCCP to the cells incubatedin the light depolarized the membrane potential (black trace).

FIG. 1. Expression of functional PR by S. oneidensis. (A) Cell pel-lets of S. oneidensis expressing PR (�) or containing an empty controlplasmid (�) grown overnight at 30°C in LB medium supplemented (�)or not supplemented (�) with retinal. Cells expressing PR in thepresence of retinal have additional red pigment. (B) Difference in theabsorption spectra of cultures of S. oneidensis expressing PR andcultures of S. oneidensis containing an empty control plasmid grownanaerobically in minimal medium containing 10 �M retinal.

VOL. 76, 2010 LIGHT-DRIVEN PROTON PUMPING IN S. ONEIDENSIS 4125

dividual cells were measured by flow cytometry (Fig. 2). Whilecultures incubated in the dark without lactate contained bothenergized cells (high fluorescence intensity) and deenergized cells(low fluorescence intensity), cultures incubated either in the darkwith lactate or in the light without lactate contained only ener-gized cells. Addition of the protonophore CCCP depolarized themembrane potential, demonstrating that PR pumps protons outof the cytoplasm to increase the membrane potential. Incubationof starved cells expressing the empty control plasmid in the lightdid not generate a population of energized cells (see Fig. S1 in thesupplemental material).

PR enhances survival during stationary phase. Althoughthe results described above showed that recombinant PR op-erates as a light-driven proton pump, it is not clear how PRinfluences the physiology of S. oneidensis. In bacteria that nat-urally contain PR, light-driven proton pumping is hypothesizedto provide a selective advantage during stationary phase (9, 10,25). To explore whether this benefit can be observed in anengineered bacterium that does not express PR naturally, wemeasured the anaerobic growth of cultures of S. oneidensisexpressing PR in minimal medium supplemented with retinalboth in the light and in the dark (Fig. 3). While the opticaldensities at 600 nm of both cultures grown in the light andcultures grown in the dark peaked at 25 h, only cultures grownin the light remained in stationary phase for �6 days with adensity of 109 viable cells ml�1. For cultures grown in the dark,the optical density decreased immediately after the maximumvalue was reached, and after 6 days the density was 106 viablecells ml�1. Regardless of illumination, the viability of controlcultures of S. oneidensis harboring an empty control plasmid orcultures of S. oneidensis expressing PR without retinal steadilydecreased after the maximum cell density was reached at 25 h(see Fig. S2 in the supplemental material).

PR enhances electricity generation. With the demonstrationthat recombinant PR is functional and integrated into thephysiology of S. oneidensis, we investigated whether light-driven proton pumping by PR can influence extracellular res-piration and generation of electricity. In a series of reactions inS. oneidensis that have not been fully elucidated, oxidation of

lactate to acetate leads to reduction of menaquinone and for-mation of biosynthetic precursors. In the presence of an elec-trode at an appropriate potential, the redox loop is completedby oxidation of menaquinol and subsequent transfer of elec-trons via several cytochromes to the outside of the cell, wheresmall-molecule mediators assist in transfer to the electrode(21, 23). Electron transfer to the electrode can be observeddirectly by measuring the oxidation current (24). After inocu-lation, cells attach to the electrode, which is a requirement forrespiration and growth because mediators do not efficientlycarry electrons from planktonic cells to an electrode (23).

Illumination of recombinant S. oneidensis cultures express-ing PR growing anaerobically on electrodes (Fig. 4A) resultedin light-dependent increases in the oxidation current (Fig. 4B).Light-dependent changes were not observed in cultures con-taining an empty control plasmid. The oxidation current mea-sured in electrochemical chambers inoculated with S. oneiden-sis either expressing PR or containing the empty controlplasmid increased for approximately 70 h as bacteria colonizedthe electrode. After reaching asymptotic values that did notdepend on expression of PR and were sensitive to conditionsduring inoculation, the current remained steady until lactatewas depleted from the medium.

The effect of light-dependent proton pumping by PR on therespiration rate of anaerobically growing cells was measured byilluminating the cultures periodically, and the sawtooth re-

FIG. 3. PR extends the viability of S. oneidensis during stationarygrowth. Cell growth was monitored by measuring the optical density at600 nm for anaerobic cultures of S. oneidensis expressing PR in thepresence of 3 mW cm�2 of light (squares) or in the dark (circles).Cultures of S. oneidensis were incubated anaerobically at 30°C in min-imal medium containing lactate, fumarate, and retinal. The values aremeans standard deviations for three independent cultures.

FIG. 4. Light-dependent current increases in electrochemicalchambers containing S. oneidensis expressing PR. (A) LED arrays usedfor illumination and glass cone of the electrochemical chamber con-taining both the working electrode and the counter electrode. So thatdetails of the interior of the chamber could be seen, it was necessary toreduce the light intensity compared to the intensity used during theexperiments and to remove reflections of the LEDs on the glass sur-faces. (B) Oxidation current of an electrochemical chamber inoculatedwith S. oneidensis expressing PR (red trace) or containing a controlplasmid (black trace). The arrows indicate the beginning of each 1-hillumination period. The light intensity was 10 mW cm�2, and thetraces are representative of three independent experiments.

4126 JOHNSON ET AL. APPL. ENVIRON. MICROBIOL.

sponse to changes in light intensity shown in Fig. 4B wasrepresentative of all experiments. Typically, after 10 min ofillumination the current increased to a value that was 0.04 0.01 A m�2 greater than the value before illumination; whenthe light was removed, the current decreased within 1 h to avalue expected for an identical experiment in which there wasno illumination. The magnitude of the light-dependent in-crease in current represented a 100% increase during earlystages of growth. As the absolute light-dependent current wasrelatively constant as more cells attached to the electrode,illumination at later stages resulted in a 30% increase in therespiration current, indicating that there may have beenchanges in light penetration for thicker biofilms.

The light-dependent increase in current was proportional tothe intensity of the light used to illuminate fully colonizedelectrodes (Fig. 5). Saturation of the light-dependent increasedid not occur with intensities of 10 mW cm�2. Previous studieshave shown that saturation of the light-dependent increases forplanktonic E. coli expressing PR (44) and halobacteria express-ing bacteriorhodopsin (13) occurs at approximately 20 mWcm�2. Addition of exogenous riboflavin, which was previouslyidentified as an extracellular electron mediator produced by S.oneidensis (23, 43), did not alter the magnitude of the light-dependent change in current.

The effects of continuous illumination on the current andmetabolite concentrations for an electrochemical chambercontaining S. oneidensis expressing PR were measured (Fig. 6and Table 1). After inoculation and attachment of cells to theelectrode, planktonic cells and the medium surrounding theelectrode were removed and replaced with fresh medium (zerotime). In the dark, the current increased as cell growth contin-ued on the electrode. After a second medium exchange andillumination of the chamber, the average rate of current pro-duction increased, and lactate was consumed more rapidly.

For both light and dark periods, decreases in lactate con-centration were matched by increases in acetate and pyruvateconcentrations. To obtain precise carbon recovery values and

to prevent complete oxidation of lactate and acetate to CO2 byaerobic respiration or consumption of H2 produced at thecounter electrode, it was necessary to sparge the chambers withN2. Under these conditions and regardless of illumination, thenumber of electrons recovered at the electrode was less thanthe expected value. As observed previously (4), it is likely thathigh N2 flushing rates decrease the H2 partial pressure andincrease the rate of hydrogenase-catalyzed oxidation of mena-quinol and reduction of protons to form H2 (26). Regardless ofthe mode of electron disposal and variations in cell growth andattachment to the electrode, illumination of PR-containingchambers was always correlated with an increased rate of ox-idation of lactate to acetate.

DISCUSSION

As evidenced by the occurrence of proteorhodopsins in en-vironments and clades of bacteria, a PR is expected to providea selective advantage. Given the role of this molecule as alight-dependent proton pump, it was hypothesized that PRcould provide an advantage under nutrient-limited conditions(9, 10). Despite attempts to measure this advantage, mostgrowth rates of bacteria expressing PR are not higher in thelight (10). We provide evidence here that PR allows cells toremain viable for extended periods. Our results not only sug-gest that PR provides a selective advantage by maintaining a

FIG. 5. Magnitude of the oxidation current depends on the lightintensity. The change in oxidation current was measured for an elec-trochemical chamber containing an electrode fully colonized (�70 h)by cells of S. oneidensis expressing PR during illumination using thefollowing 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 tostabilize before the next experiment. The arrows indicate the beginningand end of the illumination period.

FIG. 6. Current for an electrochemical chamber containing S. onei-densis expressing PR during periods of constant darkness or constantlight. After inoculation of the electrochemical chamber and attach-ment to the electrode, planktonic cells and the medium surroundingthe electrode were removed and replaced with fresh medium (zerotime). After a second medium exchange (at 67 h), the chamber wasilluminated continuously using a light intensity of 10 mW cm�2.

TABLE 1. Carbon balance for electrochemical chamberin dark and light conditionsa

Conditions Time (h)Concn (mM) of: Lactate

consumptionrate

(�mol h�1)

Avgcurrent

(A m�2)Lactate Acetate Pyruvate

Dark 0 32.0 0.0 0.467 13.7 15.3 2.5 4.1 0.15

Light 68 32.4 4.6 0.2117 6.7 25.2 0.4 7.8 0.39

a For experimental details, see Materials and Methods and the legend to Fig. 6.

VOL. 76, 2010 LIGHT-DRIVEN PROTON PUMPING IN S. ONEIDENSIS 4127

membrane potential during stationary phase but also show thatthis benefit can be transferred easily into a bacterium that doesnot contain a PR naturally, which may explain the widespreadoccurrence of this protein in many different groups of marinebacteria that live under long-term nutrient limitation condi-tions in the open ocean (38).

The results of our electrochemical studies demonstrate thatlight energy can be used by cultures of S. oneidensis expressinga light-dependent proton pump to enhance generation of elec-tric current. Compared to the time required for S. oneidensis todivide during anaerobic growth or to increase its biomass,light-induced changes in current were rapid, suggesting thatrather than altering protein expression, illumination increasedthe rates of extracellular respiration by individual cells. Be-cause all electrons deposited on an electrode must originatefrom lactate, any increase in current is proportional to anincrease in lactate consumption. Our measurements demon-strate that electrochemical chambers containing S. oneidensisexpressing PR generate more current and consume more lac-tate in the light than in the dark (Table 1).

Well-established observations have demonstrated that smallchanges in membrane potential lead to large increases in trans-port rates and accumulation ratios for small molecules (6, 7,19). Small increases in the intracellular pH also result in in-creased accumulation of weak acids, such as lactate. For ex-ample, for symport of a molecule such as lactate compensatedby one charge (32), the Nernst equation (12) predicts that a 7-to 18-mV increase should cause a 30 to 100% increase in theintracellular concentration. Similarly, 0.1- to 0.3-unit pHchanges can result in a similar accumulation ratio. Such mod-est changes in the membrane potential or pH are consistentwith the proton-pumping capacity of PR (27, 29), suggestingthat illumination leads to increased lactate uptake, increasedreduction of electron carriers such as NAD�/NADH andmenaquinone/menaquinol, and a need for faster regenerationof oxidized electron carriers (Fig. 7). A similar mechanism maybe involved in the light-induced increases in current in a pho-tosynthetic Rhodopseudomonas species isolated using amor-phous iron(III) as an electron acceptor (45).

While the increase in lactate uptake due to PR could bepredicted, the increase in the S. oneidensis respiration rate wasunexpected. Previous bioenergetic experiments with respiringE. coli and mitochondria have shown that the membrane po-tential and the rate of respiration are inversely related (5, 28).In a regulatory loop known as respiratory control, increases inthe membrane potential slow processes that transport protonsout of the cell by increasing the energy required to pumpprotons across the membrane. As electron transport is linkedto pumping of many protons (10 H� pumped for 2 e� trans-ferred from NADH to oxygen) (41), a high membrane poten-tial feeds back to limit respiration. However, if the net respi-ratory reaction is coupled to less proton pumping, respiratorycontrol would not be expected to have such a powerful influ-ence. For anaerobic growth of S. oneidensis, the 4-electronoxidation of lactate should not result in translocation of morethan 2 net protons (2 H� pumped for 2 e� transferred fromformate to menaquinone) (41), and the translocation could beless if protons are also used in import and export of solutes.The observation that S. oneidensis does not exhibit respiratorycontrol makes this bacterium an attractive platform for strat-

egies combining electron transport and proton motive forcegeneration and suggests further experiments to examine therole of the membrane potential of S. oneidensis during anaer-obic growth.

While the amount of electrical energy generated is smallerthan the amount of light energy used to illuminate the elec-trode under laboratory conditions, our work shows that it ispossible to integrate light-driven reactions into a nonphotosyn-thetic bacterium to alter survival and metabolism, with effectssuch as acceleration of electricity generation. In addition toincreasing the rates of oxidation of carbon sources, light-in-duced proton gradients incorporated into engineered bacteriamay be used to drive energy-demanding synthetic reactions forproduction of fine chemicals. More sophisticated light-convert-ing systems, such as photosynthetic reaction centers, may fur-ther improve the efficiency and yield of light energy captureand conversion.

ACKNOWLEDGMENTS

We thank Ed DeLong (Massachusetts Institute of Technology) forhis gift of the plasmid containing the proteorhodopsin gene, TonyDean (University of Minnesota) for use of the flow cytometer, andErin Marasco (Cargill, Inc.) for performing preliminary experiments.

This research was supported by the Institute on the Environment(University of Minnesota) and by National Science Foundation grantCBET-0756296.

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FIG. 7. Schematic diagram of current generation by S. oneidensisexpressing PR under anaerobic conditions with lactate as a carbonsource. 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. Oxi-dation of lactate leads to production of acetate, ATP, and reducedelectron carriers, such as NADH and menaquinol. The enzymes thatlikely are involved in reducing the electron carriers include lactatedehydrogenase, NADH dehydrogenase, and formate dehydrogenase;the reactions may also transport protons. Electrons are passed throughthe Mtr extracellular respiratory pathway to the electrode (37).

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