Proteorhodopsin (PR) is a light-powered proton pump that belongs to the rhodopsin family. It is a 7-transmembrane protein, which uses all-trans-retinal as the chromophore. It uses light energy to generate an outward proton flux. The increased proton motive force across the membrane can power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor [1]. Furthermore, it was demonstrated that light-activated proton pumping by proteorhodopsin can drive ATP synthesis as proton reenter the cell through the H+-ATP synthase complex[2].

Schematic representation of the PR gene cluster identified in clone HF10_19P19Predicted transcription terminators are indicated in red. (Four genes are for beta-carotene synthesis, blh for retinal production, and PR itself.

The sequence of our part belongs to the uncultured marine Gammaproteobacteria of the SAR86 group. The original cluster is composed of 6 genes: four are involved in beta- carotene production; one is implied in beta carotene cleavage into two molecules of retinal, the other encodes for proteorhodopsin. From the analysis of our part sequence we found out that our protein belongs to the blue absorbing group. [3]

Proposed mechanism of PR associated to the ATP-synthase complex Light-activated proteorhodopsin pumps protons outwardly, increasing the proton motive force. Protons can then reenter the cells through ATP-synthase complex, powering the ATP production.

The expected molecular size is 28 KDa. The SDS gel shows a band corresponding to around 37 KDa, as it was seen in other studies [4]. This is probably due to post-translational modifications.

Expression of ProteorhodopsinNEB10β cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 with 5 mM arabinose and 10 μM of retinal at 30°C or 37°C. Negative control were cells transformed with BBa_K731201 (i.e. araC-pBAD).

Purification of Proteorhodopsin. NEB10β cells transformed with BBa_K1604010 and BBa_K731201 were induced in LB at 37°C in the presence of retinal. The cell pellets were resuspended in 50 mM Tris-Cl pH 8 with 5 mM MgCl2 and sonicated. The lysate was centrifuged at 10,000 rpm for 20 min at 4C. The supernatant was ultracentrifuged for 100,000 g for 3 hours at 4C. The three tubes in front contain proteorhodopsin purified fractions and the three tubes in the back are negative controls treated in the same conditions

Proteorhodopsin expression in M9Cells transformed with BBa_K1604010 and BBa_K731201 were grown in LB and transferred in M9 at an OD of 0.6 and induced with arabinose with the presence of 10 µM of retinal. After 6 hours of induction the cells were centrifuged and the supernatant was discarded. From left to right: araC-pBAD induced with retinal (A), proteorhodopsin induced with retinal (B), proteorhodopsin induced (C) and not induced (D) both without retinal.

Proteorhodpsin is a light activated proton pump that exploits the conformational change of all trans-retinal to all cis-retinal. The different absorption properties are due to a single amino acid, at position 105 in the retinal binding pocket. The presence of a highly conserved Gln at position 105 in BBa_K1604010 indicates that it belongs to the blue absorbing family. [3]

Apparatus for anaerobiosis growthPanel A) sealed sterile bottles. Panel B) Anaerobic chamber.

We tested if light activation with a white light bulb (160W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria survive better anaerobically.

Anaerobiosis was achieved using sealed glass bottles with a rubber septum. We got from the local pharmacy 12 sterile bottles of physiological solution. After removing the liquid, washing them and autoclaving them, the bottles were ready to host our bacteria!

Anaerobioc growth of BBa_K1604010 E. coli transformed with BBa_K1604010 (blue line) and BBa_K731201 (green line) were grown in LB at 37°C untilan OD of 0.6 and induced in M9 minimal medium with 5 mM arabinose and 10 uM retinal in the dark. After 5 hours of induction the culture were transferred in sealed bottles in the anaerobic chamber and placed again in the thermoshaker. Sample in the dark were kept in aluminum foil. Light exposed samples were excited with a 160W halogen light bulb placed outside the incubator. The blue line (proteorhodopsin) is the result of the average of 6 different samples (3 in the dark and 3 in the light) while the green line (araC-pBAD) is the average of 1 sample in the dark and 1 in the light.

After five hours of induction in the dark (i.e. the samples were wrapped in aluminum foils) the cultures were split in the anaerobic chamber in light and dark conditions. The cultures were placed in the thermoshaker that was illuminated from the outside. Half of the cultures were kept in the dark and the other half were exposed to the light.
The OD₆₀₀ was constantly monitored because E. coli’s growth is slowed down in stressful conditions such as the lack of oxygen.

The bacteria expressing proteorhodopsin have an increased lifetime when compared to a negative control with araC-pBAD (BBa_K731201). However we did not observe significant changes between light and dark with this test. The explanations could be several. Most likely we were not exciting properly the system. However it seems that there is a basal functionality even in the absence of light, probably due to activation of the proton pump independently from light exposure.

While we decided to explore different light sources, we built a solar mimicking apparatus, that would allow us to directly illuminate the samples without the glass of the thermoshaker.

Solar mimicking apparatus NEB10β cells transformed with BBa_K1604010 were grown exposed to light (left side) or in dark condition (right side). The cultures were maintained at ~37°C with magnetic stirring using a laboratory plate. The light was provided by a 160 Watt halogen lamp placed 4 cm from each culture. The dark condition was simulated covering the cultures with aluminum foil (not shown).

Since we observed that there was a possible activation of the proton pump without light, we decided that our next test would be a proton pumping experiment as described in the literature [2][5].

The ΔpH between the light exposed proteorhodopsin and the two negative controls (proteorhodopsin in the dark and araC-pBAD in the light is 0.22. This result evidenced that although there is a basal acidification of the medium due to the bacteria metabolism, our device acidifies the medium thank to the activation of the proton pump when the bacteria were light exposed.

We improved the proteorhodopsin part that we extracted from the registry, placed under an inducible promoter and we fully characterized it to demonstrate that the proton pump doeswork when the bacteria are light exposed. This membrane protein does require retinal to properly fold and increases the lifespan and the vitality of the engineered bacteria in anaerobic conditions. We did experience some difficulties in finding the right conditions of growth, light exposure and to reach anareobiosis. Also from our experience, this is a delicate system that showed sometime variability in the measurements between different biological samples. However we optimized the system and we now have a functioning device that can be used in our MFC.

Proteorhodopsin are happy to stay in our Microbial fuel Cell under the sun. Check out our results. [link alla pag della MFC]