Temperature Dependency of Proton Pumping Activity for Marine Microbial Rhodopsin from Antartic Ocean

Proteorhodopsin (PR) is discovered from marine bacteria and it has proton pumping activity from inside to outside of the cell using light energy. In general, PR classified into two groups by the maximum absorption spectra. In this study, we isolated the two of a full sequence of opsin homologues by PCR from the seawater sample near King George Island, Antarctica. One was the same sequence as the first reported GPR (Green-light absorbing PR) from Monterey Bay. Another named HSG119 was a newly discovered sequence which shows high sequence similarity with BPR (Blue-light absorbing PR). HSG119 has an absorption maximum at 493 nm with broader spectrum at pH7.0 and it can pump protons out of the cell membrane. Interestingly, it showed a similar temperature dependence to GPR(Y200N) that isolated near the North pole.

Central North Pacific Ocean have green absorption maxima (525 nm) at pH7.0, whereas HOT_75m4 from the same place in deep-sea have blue absorption maxima (490 nm) at pH7.0 8 . The depth of host living brings the difference between those groups. It is primarily related to light penetration availability upon a proportion of the depth. Surface waters are exposed to white light, whereas deeper ocean is mostly exposed to the blue light. It is due to stronger scattering and absorption of other wavelength light at the upper depth 13 . Also, several amino acids changes on retinal binding pocket trigger dramatic change of absorption maximum 20 . If 105th position on PR (Leu in GPR and Gln in BPR) is changed to each other, their absorption spectra also can be interconverted. To adapt to cold environmental temperature, the photo cycling rate of PRs was modified. NPR8 from the sea near the Korea Arctic Research Station Dasan at NyAlesund, Svalbard, Norway showed a different temperature dependency between GPR and BPR 21 .
Interestingly, only GPR type PRs were found at the Arctic Ocean, but GPR and BPR types were identified on the surface from the Antarctic ocean. For this study, we isolated new BPR type gene from Antarctic Ocean and it was characterized by using several biophysical methods such as light-driven proton pumping assays, and absorption spectroscopy, laser flash-induced photolysis technique.

Results
two pR homologue genes were founded in total genomic DnA samples from sea near research station King SeJong. Proteorhodopsin homologues genes were amplified from each genomic DNA samples by PCR using conserved primers and degenerate primers (Table S1). The degenerate primers were designed on the basis of the conserved N-terminal and C-terminal regions of previously studies 10,15 . The conserved primers were designed on the conserved regions on helix C and F. We found two PR full length DNA sequences from metagenome sample CKH120(CKH070120-01) and HSG119(HSG070119-03). Genes from CKH120 are perfectly identical to GPR. However, the genes from HSG119 showed several differences on primary sequence. HSG119 showed 99% amino acid sequence homology to BPR and 80% to GPR. Along the amino acid sequence alignment, HSG119 has 3 different positions to BPR (Fig. 1).
Photochemical properties of HSG119 from antarctic ocean. Absorption maxima of HSG119 rhodopsin is 535 nm, 493 nm, and 490 nm at pH 4, 7 and 10, respectively (Fig. 2). It is very similar spectra to that of BPR. But, there are some shoulders on both side of HSG119 rhodopsin spectrum at pH 7. When the proteins are under pH10, right side shoulder is disappeared but left side shoulder is still existed. Both shoulders are disappeared if the proteins are under pH 4. Absorption spectrum is shifted by the proton concentration. Especially, it showed large red shift (39 nm) at pH 4 versus spectrum at pH 7. We collected absorption spectra of HSG119 rhodopsin under various pH conditions. We could titrate the Schiff base counter-ion of HSG119 rhodopsin from spectra under various pH conditions. Only one isosbestic point is shown at around 500 nm wavelength ( Fig. 3 upper picture). We used absorption maximum at the 550 nm and set spectrum of pH 7 as a reference for calculation. After the calculation, we could get the pKa value of HSG119 rhodopsin around 5.8 and 8.0. Observation of two pKa is similar feature of previous reported BPR (pKa at 6.2 and 7.8)* (Fig. 3 lower picture). Before functional study for HSG119 rhodopsin, we measured photocycling rate for measuring photointermedates. First, we could detect O intermediates by the time-resolved difference spectra measurement (Fig. 4). There is negative direction spectrum change between 450 and 490 nm wavelength when protein got light activation. It is broad, but the absorption spectrum of HSG119 rhodopsin (at pH 10) is near at that range. There is a big positive peak of O intermediate around at 550 nm wavelength. The amplitude of peak is very similar as negative peak around at   www.nature.com/scientificreports www.nature.com/scientificreports/ 480 nm by decrease of ground state. We measured the spectral changes for 15 sec after light off. M intermediate of HSG119 could not be measured and O decay is slower than GPR and BPR, but faster than NPR8 that contains Y200N mutation 21 .
Temperature dependency of HSG119 rhodopsin from antarctic ocean. We measured photocycling rate of HSG119 rhodopsins using various temperature. We focused on temperature dependency of O decay of HSG119. We set the temperature conditions as 5 °C, 15 °C, 25 °C and 35 °C (Fig. 5). HSG119 rhodopsin shows positive relationship on temperature and photocycling rate. We compared temperature dependency with GPR, BPR and NPR8 (Y200N) based on the slopes of the Arrhenius plot (Fig. 6). NPR8 was well-studied in previously report, the slope of NPR8 is in the middle between those of GPR and BPR.. Temperature dependency of HSG119 is similar to that of NPR8 (Y200N) than that of GPR and BPR 21 . When temperature drops, the photocycling rate of O intermediates of HSG119 also decreased down Proton pumping activity of HSG119 rhodopsin. For the functional study, we measured proton pumping ability (Fig. 7). We made sphaeroplast of HSG119 expressed cell. First, we put sphaeroplast at dark condition for 1 min. After dark adaptation, we measured the pH changes of 1 min duration of dark and light illumination. HSG119 rhodopsin clearly showed outward proton transfer during light activation. It is pretty good efficiency compared to almost 60% level of GPR.
Optical property of HSG119 mutants, D97N and E108Q. Two important residues of HSG119 rhodopsin are verified by characterization of mutants -the proton acceptor Asp97 and donor Glu108, respectively. We constructed D97N and E108Q mutants. We checked absorption spectra for each mutant. Both D97N and E108Q showed large red shift compare to WT of HSG119 rhodopsin. Each mutant absorbs light maximally at 535 nm (D97N HSG119) and 529 nm (E108Q HSG119). Shoulders on both side of WT were no longer existed in D97N mutant. Otherwise, E108Q still has broad shoulder on left side which of the amplitude is larger than WT. But right shoulder was disappeared (Fig. 8). www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
PRs from Arctic and Antarctic Ocean are well-known in terms of their sequences and absorption maxima 8,13,14,22 . In the previous study, PalE6 (southern ocean Palmer station: PAL clone) that was reported from the Antarctic Ocean absorbs blue light (λmax = 490 nm, pH 7). However, most of the PR variants in the Arctic Ocean absorb green light (λmax = 527 nm, pH 7). Their amino acid sequences are similar to that of GPR from Monterey Bay Proteorhodopsin 8 . Based on the previous report, the color of PRs has a small influence on ion transport activity 21 .
In this study, we found BPR homologue HSG119 rhodopsin from the Antarctic Ocean. HSG119 rhodopsin has three different changes upon BPR. HSG119 has maximum absorption at 493 nm at pH 7 that is similar to  www.nature.com/scientificreports www.nature.com/scientificreports/ BPR, but it did not show the typical spectrum shape of type I rhodopsin. HSG119 rhodopsin has two shoulders on both sides of the spectrum. These shoulders make HSG119 absorbs light broadly and not only blue light but also green light region. Compared with BPR, GPR and HSG119 rhodopsin at 525 nm, absorption maxima of GPR, HSG119 rhodopsin absorbs light above 80% of GPR and also over 10% than those of BPR. We constructed two mutants D97N and E108Q, those were mutations of proton acceptor and donor, respectively. At D97N mutant lost its shoulders and therefore it is proved that the protonation of proton accepter is involved in its shoulders. Probably, these shoulders were influenced by the interaction between the negative charge of Asp97 and chromophore, retinal. Also, HSG119 rhodopsin has methionine at 106 position, but BPR has valine and this position is a nearby retinal binding site that might involve in color-tuning.
Previously, it is suggested that PRs could be divided based on the photocycling rate rather than absorption maxima because the absorption peak of PRs has little relationship with their function, proton transport. NPR8 showed the dependency on temperature between those of GPR and BPR. We have reported that Arctic PRs have adapted to environmental temperature changes 21 . Here we showed that the temperature dependency of HSG119 rhodopsin was similar to NPR8 as expected. The big difference of HSG119 rhodopsin compared to NPR8 is proton pumping activity. HSG119 has good proton pumping activity but NPR8 does not has it. HSG119 rhodopsin could chase two hares called cold adaptation and proton pumping activity at once and successfully adapted.  www.nature.com/scientificreports www.nature.com/scientificreports/ The result showed on cold adaptation of Arctic PRs cannot be answered for the distribution of PRs. The color of PRs has little related to cold temperature adaptation. Here, we suggested the answer for why the BPR like rhodopsin existed on the surface of Antartic Ocean. Metagenome samples were collected from Marian Cove, inside of Maxwell Bay. The glacial wall of this area collapses very often. Also, deep ocean heat melting the Antarctica's ice. Because of that, sediment, many organic nutrients and materials are mixed easily and often in this area. This phenomenon caused the surface of the sea to be quite turbid 23 . And the range of tide in this area is 1,800 mm. It makes frequent seawater currents. Rising current also can be generated and it caused the migration of middle or deep-sea microbe to the surface of the sea. These might be able to make the finding of BPR like rhodopsins at the sea surface area.
Briefly, these results suggested that the protein activity is modified and adapted if environmental conditions are changed.

Methods
Collection of samples and extraction of DNA from the marine bacteria. The Antarctic samples were collected from sea surface from the region around King Sejong Station at King George Island (62°13.208′S -58°47.114'W), Antarctica (Table S2). Water temperature was 0.9 °C, pH was 7.74, and salinity was 32.6‰. Two liters of seawater were concentrated using 0.2 μm Sterivex filters (Millipore) and transported to the laboratory in Korea at −20 °C. Total genomic DNA was extracted using a modified CTAB method as described in the previous study 24 .
Measurement of the absorption spectra and pKas. The setting for absorption spectroscopy to measure absorption maxima of the the rhodopsin and pKas of the Schiff base counterion in purified PRs is followed to previous research 21 . The absorption spectra were recorded with UV/VIS spectrophotometer (UV-2550, Shimadzu, Japan) at pH 4, pH 7.74, and pH 10. In order to calculate the pKas of the primary proton acceptor, performed the pH titration in the range of pH 4 ~ pH 10 and the spectrum at pH 7.0 was used as a reference. Collected data from different absorption spectra were determined and fitted with functions containing pKa components[y = A/ (1 + 10 pH-pKa )] using Origin Pro7.0 26 . preparation of the sphaeroplast and proton pumping measurements. To measure the pumping activity, the sphaeroplast of new isolated PR is prepared similar to previous research 21,26 . Rhodopsin-expressed cells (250 mL) were centrifuged and suspended in 10 mL of plasmolysis buffer (30 mM Tris-HCl, pH 8.0, and 20% sucrose) with lysozyme (Usb, USA). Sphaeroplasts were collected and resuspended in 400 uL DNA lysis buffer (100 mM KPi, pH 7.0, 20 mM MgSO 4 , 20% sucrose, 1.6 mg DNase I) and injected using a 1 mL syringe (26 gauge needle) into 200 mL of rapidly shaking osmotic shock solution (50 mM KPi, pH 7.0). After shaking at 37 °C for 15 min, Na-EDTA was added to a final concentration of 10 mM and shaked for 15 min, MgSO 4 was added to a final concentration of 15 mM and shaked for another 15 min. Sphaeroplast vesicles were collected at 30,778 × g for 1 hr at 4 °C (Beckman XL-90 ultracentrifuge, USA) and washed with 10 mL unbuffered solution (10 mM NaCl, 10 mM MgSO 4, 10 uM CaCl 2 ). Finally, sphaeroplasts were resuspended in 3 mL of unbuffered solution 26 . Samples were illuminated through the short wave cut-off filter (>440 nm, Sigma Koki SCF-50S-44Y, Japan) in combination of focusing convex lens and heat-protecting (CuSO 4 ) filter. The pH values were monitored (Horiba pH meter F-51) and pH data were transferred and recorded automatically with Horiba data Navi program. Starting pH was pH 8.2. After one minute for dark adaptation, one minute for dark and one minute for white light illumination were repeated for 4 times.

time-resolved difference spectra and laser induced absorption difference spectroscopy.
Samples were illuminated through Cole-Parmer illuminator (41720 series) and time-resolved difference spectra were measured by S-3100 diode-array spectrophotometer (Shinco, Korea). Flash-induced absorbance changes