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Appl Microbiol Biotechnol (2013) 97:819–828 DOI 10.1007/s00253-012-4452-y METHODS AND PROTOCOLS Characterization of the chimeric seven-transmembrane protein containing conserved region of helix C–F of microbial rhodopsin from Ganges River Ah Reum Choi & Se Jun Kim & Byung Hoon Jung & Kwang-Hwan Jung Received: 20 March 2012 / Revised: 9 September 2012 / Accepted: 19 September 2012 / Published online: 15 November 2012 # Springer-Verlag Berlin Heidelberg 2012 Abstract Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria. Recently, many PR-like genes were found in non-marine environments. The goal of this study is to explore the function of rhodopsins that exist only as partial proteoopsin genes using chimeras with marine green PR (GPR). We isolated nine partial genes of PR homologues using polymerase chain reaction (PCR) and chose three homologues of GPR from the surface of the Ganges River, which has earned them the name “CFR, Chimeric Freshwater Rhodopsin.” In order to characterize the proteins, we constructed the cassette based on GPR sequence without helices C to F and inserted the isolated conserved partial sequences. When expressed in E. coli, we could observe light-driven proton pumping activity similar to proteorhodopsin, however, photocycle kinetics of CFRs are much slower than proteorhodopsin. Half-time decay of O intermediates of CFRs ranged between 143 and 333 ms at pH 10; their absorption maxima were between 515 and 522 nm at pH 7. We can guess that the function of native rhodopsin, a retinal protein of fresh water bacteria, may be a light-driven proton transport based on the results from chimeric freshwater rhodopsins. This approach will enable many labs that keep reporting partial PCR-based opsin sequences to finally characterize their proteins. Keywords Opsin . Membrane protein . Proton pumping . Environmental genomics . Freshwater rhodopsin A. R. Choi : S. J. Kim : B. H. Jung : K.-H. Jung (*) Department of Life Science and Institute of Biological Interfaces, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, South Korea e-mail: [email protected] Introduction Proteorhodopsin (PR) was first discovered 10 years ago by Beja et al. (Atamna-Ismaeel et al. 2008) in uncultivated marine γ-proteobacteria of SAR86 group (Beja et al. 2000, 2001; Brown and Jung 2006). It has been shown that PR is a type I rhodopsin which functions as a light-driven proton transporter using retinal photo-isomerization and subsequent protein conformational changes. Since then, a large number of PR homologues were found in bacteria throughout the marine photic zone such as Monterey Bay (Eastern Pacific Ocean), Hawaii Ocean Time (HOT, Central North Pacific Ocean), Antarctic Peninsula, Mediterranean Sea, Red Sea, Sargasso Sea, Arctic Ocean, and Antarctic Sea ice (Beja et al. 2001; de la Torre et al. 2003; Frigaard et al. 2006; Jung et al. 2008; Koh et al. 2010; Man et al. 2003; Man-Aharonovich et al. 2004; McCarren and DeLong 2007; Sabehi et al. 2005, 2003; Venter et al. 2004). Recently, it has been reported that the genes of the archaeal-type rhodopsins are present in some non-marine bacteria (Sharma et al. 2008). More divergent proteorhodopsin-related sequences originating from non-marine organisms like Gloeobacter violaceus (Nakamura et al. 2003) and Roseiflexus sp. RS-1 (Hanada et al. 2002), bacteria isolated from a calcareous rock, and a Japanese hot spring have been found as well. Gloeobacter rhodopsin is characterized as a fast-cycling rhodopsin capable of light-driven proton transport, similar to proteorhodopsin (Miranda et al. 2009). Similarly, many actinorhodopsin genes, proteorhodopsin-like sequences, were found predominantly in non-marine environments (Sharma et al. 2009, 2008). The discovery led to novel understanding of survival strategies and the role of phototrophy in biogeochemical cycles (de la Torre et al. 2003; Fuhrman et al. 2008). Extensive insight was gained into the evolutionary relationship between different photosystems (Frigaard et al. 2006; McCarren and DeLong 2007; 820 Sharma et al. 2006; Spudich 2006) and biogeographical analyses that have shown important differences in PRs in different geographical and ecological contexts (Papke et al. 2003; Pasic et al. 2005; Sabehi et al. 2003). Many PRs and their homologues have been discovered by polymerase chain reaction (PCR)-based gene survey using degenerate primers, or through genome sequencing of bacterial artificial chromosome, fosmids, and with environmental shotgun libraries (Beja et al. 2001; de la Torre et al. 2003; Frigaard et al. 2006; Jung et al. 2008; Koh et al. 2010; Man et al. 2003; Man-Aharonovich et al. 2004; McCarren and DeLong 2007; Sabehi et al. 2005, 2003; Venter et al. 2004). The absorption maxima of PR variants depend on the places and depth of the ocean where their hosts reside (Beja et al. 2001; Man et al. 2003). PR variants from the surface or the deep ocean from the same place (e.g., Hawaiian Pacific Ocean) have different absorption maxima being spectrally tuned to usable light in their environment (Beja et al. 2001). PR from Monterey Bay and HOT_0m (surface) has absorption maxima tuned for green light (525 nm), whereas PRs from the Antarctic Ocean and Hot_75m4 (75 m deep) have blue-shifted absorption maxima (490 nm). This is achieved by a substitution of a single amino acid residue at the position 105 (Leu in green light-absorbing proteorhodopsin (GPR) and Gln in blue light-absorbing proteorhodopsin (BPR)), which thereby functions as a spectral tuning switch (Beja et al. 2001; Man et al. 2003; Sabehi et al. 2007). There are major differences between GPR (MBP) and BPR (Hot_75m4) including the absorption maxima, photochemical reactions, and proton pumping efficiency (Chomczynski and Sacchi 1987). The natural occurrence and vertical distribution of green- versus blue-light-absorbing PRs in the oceans can therefore at least partly be accounted for by adaptation to the prevailing light spectra (Pasic et al. 2005; Wang et al. 2003). In this work, we have used degenerate PR gene primers (Rusch et al. 2007) to positively identify PR-bearing operational taxonomic units from the surface of the Ganges River. We constructed a cassette with GPR sequence missing helices C to F and inserted the found fragments in it. In addition to chimeric proteins (named CFR, Chimeric Freshwater Rhodopsin), modified GPR and chimeric GPR/ BPR (as controls) were characterized by biophysical methods such as absorption spectroscopy, light-induced difference spectroscopy, flash-induced photolysis, and lightdriven proton pumping assays. Materials and methods Sampling and extraction of total DNA A water sample (1 L) from the surface of the Ganges River in the city of Varanasi, India (25°16′55′′N, 82°57′23′′E), was Appl Microbiol Biotechnol (2013) 97:819–828 collected in December 2008 and processed immediately in Seoul, Korea. The water sample was filtered first through a 10-μm-pore-size filter and through a 0.2-μm-pore-size filter. Total genomic DNA was extracted from each filtrated fraction using the Trizol methods (Chomczynski and Sacchi 1987). PCR amplification from total genomic DNA from Ganges River, cloning, and DNA sequencing For detection of PR homologue genes in environmental genomic DNA samples from the filtrate, a multiplex PCR analysis with degenerate primers was performed. Primers were designed using conserved helix C and helix F regions of GPR. Primers used in this study are listed in Table 1. PCRs for PR homologues were performed using Taq DNA polymerase (Vivagen, Korea) in twice. PCR amplification was carried out with a total volume of 25 μl containing 1 μl (∼1 to 5 ng/μl) of template DNA, 200 μΜ deoxynucleoside triphosphates (dNTPs), 1.5 mM MgCl2, 5 pmol primers, and 0.5 units of Taq DNA polymerase. The amplification comprised the following program: an initial step at 95 °C for 1 min and then 40 cycles at 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 2.5 min. At the end of every PCR, a post-elongation step at 72 °C for 5 min was carried out. PCR products were visualized by gel electrophoresis. The product (330 bp) was excised from the gel and purified with the Qiagen gel extraction kit (Qiagen, Germany). The purified DNA fragments were cloned with the T-Blunt PCR cloning kit for DNA sequencing (SolGent, Korea) and sequenced. Design of chimeric proteins To express the full protein from the partial PR homologue sequences, we designed a cassette for chimeric rhodopsin. Table 1 Proteorhodopsin primers used in this study Primer Direction Sequence (5′–3′) (A)RYIDW (C)RYIDW LRYIDW LRYVDW FRYIDW LRYVDWILT Forward Forward Forward Forward Forward Forward GWAIYP WFLLVGWAIYP Reverse Reverse GWVIYP GWSIYP Reverse Reverse AGNTAYATHGAYTGG CGNTAYATHGAYTGG CTCCGTTATATHGAYTGG CTCCGTTATGTTGATTGG TTNMGNTAYATHGAYTGG CTCCGTTATGTTGATTGGA TTTTAACA CGGGTAAATCGCCCAACC CGGGTAAATCGCCCAACC AACTAGAAGGAACA NGGRTADATNACCCANCC NGGRTADATNSWCCANCC Parenthesis for the first two amino acids means dNTP of start Reported in Sabehi et al. 2007 Appl Microbiol Biotechnol (2013) 97:819–828 The pKA001 plasmid template contains a partial proteorhodopsin gene with N-terminal and C-terminal regions (Fig. 1). At the RYIDW region of helix C, we modified the DNA sequence to insert KpnI restriction enzyme site by site-directed mutagenesis. As a result, the RYIDW region is changed to the RYLDW. In the other conserved region, GWAIYP, the NgoMIV restriction enzyme site was created by site-directed mutagenesis from (604th bp) GTAGGT to GCCGGC (Fig. 1). Then, helix C–F regions of PR homologue sequences, BPR, BR, and NpSRII were cloned into this cassette. Expression and purification of CFRs To express chimeric fresh water rhodopsins, we transformed the plasmid into Escherichia coli UT5600 strain. The pKA001 plasmid contains a chimeric freshwater rhodopsin gene. The transformed cells were induced with 1 mM IPTG (Applichem, USA) and 5 μM all-trans retinal (Sigma, USA) for 6 h at 35 °C. The collected cells were sonicated (Branson sonifier 250), and the membrane fraction was treated with 1 % n-dodecyl-β- D -maltopyranoside (DM) (Anatrace, USA), and the protein fraction was bound to the Ni+2– NTA resin and eluted with 0.02 % DM and 250 mM imidazole (Sigma, USA). Absorption spectroscopy and pKa measurements Absorption spectroscopy was used to measure absorption spectra and to calculate pKa values of the Schiff base counterion in purified chimeric freshwater rhodopsins. The absorption spectra were recorded with Shimadzu UV-vis spectrophotometer (UV-2550) at pHs 4, 7, and 10. In order to calculate the pKas of the primary proton acceptor, the spectrum at pH 7.0 was used as a reference, and pH was lowered from 7.0 to 4.0 and raised from 7.0 to 10. The corrected ratio of protonated and deprotonated forms at different pH values was determined as previously described 821 (Wang et al. 2003) from the intensities of the absorption band that appears at λmax of each new component as the pH is changed. The data were fitted to functions containing titration components (y0A/(1+10pH−pKa)) using Origin Pro 6.1 (Wang et al. 2003), where A represents the maximal amplitude of relative absorbance changes. Proton pumping measurement Spheroplast vesicles as described (Wang et al. 2003) were isolated by centrifugation at 30,000×g for 1 h at 4 °C (Beckman XL-90 ultracentrifuge) and washed with 3 ml of 10 mM NaCl, 10 mM MgSO4·7H2O, 100 μM CaCl2 (Wang et al. 2003). Samples were illuminated at 100 W/m2 intensity through the short-wave cutoff filter (>440 nm, Sigma Koki SCF-50S-44Y, Japan) in combination with focusing convex lens and heat-protecting (CuSO4) filter, and the pH values were monitored by Horiba pH meter F-51. Light and laser-induced absorption difference spectroscopy Light-induced static absorbance changes were measured on Schinco (Korea) spectrometer. Flash-induced transient absorbance changes were measured for 10,000 ms on RSM 1000 (Olis, USA) spectrometer. The actinic flash was from an Nd-YAG pulse laser (Continuum, Mini-light II, 532 nm, 6 ns, 25 mJ). Thirty signals were averaged for measuring the rate of formation and decay of the photo-intermediates. To measure a clear signal purified membranes were incorporated into 7 % poly-acryl amide gels, which were soaked in 50 mM Tris, 150 mM NaCl at pH 10.0 (Jung et al. 2008). Nucleotide sequence accession numbers Partial opsin sequences obtained in this study were deposited in GenBank under the following accession numbers: JX169790-JX169798. Results Chimeric freshwater rhodopsin variants from the Ganges River Fig. 1 The schematic for the cassette containing N-terminal and Cterminal parts of GPR, with the topological model of the putative protein product shown at the bottom. Freshwater rhodopsin partial gene was inserted into the plasmid at the KpnI and NgoMIV restriction sites A total of nine distinct chimeric fresh water rhodopsin sequences were obtained from 96 of clone samples from the Ganges River by PCR-based gene survey. We amplified genomic DNA using the ten PR primer combinations recommended previously by Atamna-Ismaeel et al. 2008. In all primer combinations (Table 1), these sets ((A)RYIDW, (C) RYIDW, LRYIDW, LRYVDW, FRYIDW, LRYVDWILT as forward primer and GWAIYP, WFLLVGWAIYP, GWVIYP, GWSIYP as reverse primer) were used to amplify the 822 Appl Microbiol Biotechnol (2013) 97:819–828 Fig. 2 Phylogenetic tree of partial fresh water genes in Ganges River based on neighbor-joining analysis of translated nucleotide sequences. The helix C to F fragments corresponding to freshwater opsin were used for analysis. Analysis was conducted by using the sequence alignments by Clustal W. Sequences are labeled with NCBI database accession numbers homologues of PR. Pairs of primer (LRYVDW + GWAIYP, LRYVDWILT + GWAIYP, LRYVDW + WFLLVGWAIYP, and LRYVDWILT + WFLLVGWAIYP) gave positive results. Phylogenetic analysis showed that the freshwater rhodopsin sequences clustered within groups (Fig. 2). As shown in Fig. 2, each CFR of our clones is the individual sequence rather than clusters like putative microbial rhodopsin from lake Kinneret, Israel, putative proteorhodopsins from Atlantic and Pacific oceans, Antarctic Sea ice, and marine flavobacteria. The amino acid sequence alignment shows that nine partial opsin sequences differ from each other at 60 positions out of 112 residues of the cloned fragment (Fig. 3). Among these, 39 positions were located Helix C Helix D in the helices E and F, and three positions are in the retinal binding pocket. Spectral properties of the CFR/GPR and BPR/GPR chimera In this study, we have expressed the three of CFR/GPR and BRP/GPR chimera in E. coli, followed by solubilization with DM and purification through Ni +2–NTA column. First of all, the purified modified GPR possesses the λmax identical to that of the wild-type GPR (522 nm), as shown for the case of GPR whose gene contains the KpnI/NgoMIV restriction enzyme sites in Fig. 4. This is also the case for BPR/GPR (Fig. 4), which shows the identical λmax with the Helix E Helix F * * * * * * * * ** ** * CFR5 LRYVDWILTVPLMCVEFYLITKKAGGKKVLLWQLIFASLVMLVTGYIGEAIYGKESQSWIWGLISGLAYFYIVYLIWFGDVAKLAGNAGPAVQKAVKSLGWFLLVGWAIYP CFR7 LRYVDWILTVPLMCVEFYLITKKAGGKKVLLWQLIFASLVMLVTGYIGEAIYGKESQSWIWGLISGLAYFYIVYLIWFGDVAKLAGNAGPAVQKAVKSLGWFLLVGWALYP CFR9 LRYVDWILTVPLMCVEFYLITKKAGGKKVLLWQLIFASLVMLVTGYIGEAIYGKESQSWIWGLISGLAYFYIVYLIWFGDVAKLAGNAGPAVQKAVKSLGWFLLVGWAIYP CFR3 LRYVDWILTVPLMCVEFYLITKKAGGKKVLLWQLIFASLVMLVTGYIGEAIYGKESQSWIWGLISGLAYFYIVYLIWFGDVAKLAGNAGPAVQKAVKSLGWFVLVGWAIYP CFR2 LRYVDWLLTVPLMCVEFYLITKKVGSTQSLLWKLIAASVGMLVTGYVGEAIYPTESVSWVWGAISGLFYFYIVYLVWFGEVAKLAGNAGPDVAAANKTLAWFVLVGWAIYP CFR4 LRYVDWLLTVPLMCVEFYLITKKSG-GTTGLLCKMILASVVMLVTGYWGEAGLGN--ATIWGTISAIAYFYIVYEVWMGDVKKLATSAGSAVADANSALGWFVLVGWAIYP CFR8 LRYVDWLLTVPLMCVEFYLITKKAGGTIGLLWKLIIASIFMLVTGYIGEAMHGQDASSWVWGTISSIGYAYIVWLVWAGDVAKLAKSSSPAVAAANRYLGWFVLVGWAIYP CFR1 LRYVDWLLTVPLMCVEFYLITKKAG-AKTSLLWKLILASVVMLVTGFFGEATDRGN-SVLWGVISGAAYFYIAYLVWFGEVASLSNTAGPSVAKATRILAWFVLVGWAIYP CFR6 LRYVDWILTVPLMCVEFYLILKVAG-AKQSLMWKMIILSLVMLVTGYAGETIDRPN-AWLWGLISGIAYFVIVYEIWLGEASKIAQAAGGNVLSAHKILCWFLLVGWAIYP GPR 93 FRYIDWLLTVPLLICEFYLILAAATNVAGSLFKKLLVGSLVMLVFGYMGEAGIMAAWPAFIIGCLA--WVYMIYELWAGEGKSACNTASPAVQSAYNTMMYIIIFGWAIYP 201 BPR 93 FRYIDWLLTVPLQVVEFYLILAACTSVAASLFKKLLAGSLVMLGAGFAGEAGLAPVLPAFIIGMAG--WLYMIYELYMGEGKAAVSTASPAVNSAYNAMMMIIVVGWAIYP 201 Fig. 3 Amino acid sequences comparison of nine partial freshwater opsin genes from the Ganges River, GPR, and BPR. Differences between the freshwater opsins are marked with black boxes. Based on BR, transmembrane helices and residues in contact with the ** chromophore retinal are marked with black arrows and asterisks, respectively. Underlined amino acids are primer sequences. This alignment was made using Clustal W program Appl Microbiol Biotechnol (2013) 97:819–828 823 Fig. 4 Absorption spectra of GPR (w/KpnI and NgoMIV), BPR/GPR, CFR1/GPR, CFR2/GPR, and CFR3/GPR chimeras at different pH values. Purified rhodopsins were in 50 mM Tris–HCl (pH 7), 150 mM NaCl, and 0.02 % DM Table 2 The absorption maxima, the major pKas of the Schiff base counterion, and the photocycle kinetics of CFRs Name GPR w/KpnI, NgoMIV BPR/GPR CFR1/GPR CFR2/GPR CFR3/GPR λmax (pH 4) λmax (pH 7) λmax (pH 10) pKa M decay (ms) O decay (ms) 541 515 545 536 535 522 491 522 520 515 519 491 513 513 511 7.6 5.8 6.9 6.1 5.7 36 ND 352 130 130 49 70 263 119 123 The M decays and O decays were fitted to bi-exponential functions and only the rate of major component is shown ND indicates that the photocycle could not be measured due to the limitation of the instrument 824 wild-type BPR (491 nm) at pH 7. The absorption maxima of all the CFR isolates were between 515 and 522 nm at pH 7 (Table 2), which falls into the green light region. CFR 1 absorbs light maximally at 545 and 513, at pH 4 and 10, respectively (Fig. 4). The most blue-shifted CFR variant, CFR3, displayed λmax 0535 nm at pH 4 and 511 nm at pH 10 (Fig. 4). The colors of three CFRs isolates at neutral pH are illustrated in Fig. 4. CFRs were analyzed in this study contained non-polar methionine at position homologous to 105, similar to GPR with leucine at this position but unlike BPR which has polar Gln (Fig. 3), suggesting that they are green-absorbing rhodopsins. Accordingly, the absorption spectra suggest that all CFR isolates from the Ganges River absorb mainly green light. In the case of BR/GPR and NpSRII/GPR, it could not form a pigment with retinal. Counterion titration and proton pumping activities The artificial purified CFR variants were titrated to determine the pKa of the major spectral transition corresponding to the deprotonation of the Schiff base counterion, and only a major pKa is shown in Table 2. The titration of each rhodopsin did not fit well to a single pKa, presenting major and second minor components (Fig. 5). Especially in case of CFR2, it looks like two almost equal components in Fig. 5. Among them, the major component was selected by more fraction value into the Table 2. The pKa values of Asp-97 for CFRs in DM were 5.7∼6.9, in case CFR2, which is about one unit lower than that of GPR (7.6). We made right-side-out vesicles of E. coli (Wang et al. 2003) that contained each CFR for measuring proton transport activity. Pumping activity of each CFR was verified by measuring pH difference between the spheroplast suspension in the presence and absence of illumination through short-wave cutoff filter (>440 nm), where the difference of pH could be converted to the number of transported protons. CFRs have ability to translocate protons in E. coli spheroplast upon illumination (Fig. 5). At similar pigment concentration of GPR (w/KpnI, NgoMIV), three CFR/GPR chimeras, and BPR/GPR chimera, the pumping activity of CFRs is similar to one of GPR. As expected, the pumping activity of BPR/GPR chimera is lower than that of GPR, similar to the wild-type BPR (Wang et al. 2003). Photochemical properties of the CFR/GPR and BPR/GPR chimeras PR has a unique photocycle that is comprised of a series of intermediates, such as blue-shifted M and red-shifted Ointermediates. To study the photocycle of CFRs, lightinduced UV-vis spectroscopy and flash photolysis were used (Fig. 6). First, we tested if the photocycle is modified Appl Microbiol Biotechnol (2013) 97:819–828 by the introduction of KpnI and NgoMIV restriction enzyme sites. The photocycle of modified GPR with two restriction sites is similar to that of the wild-type GPR. Also, it should be noted that BPR/GPR exhibits the photocycle similar to the wild-type BPR. The left panel in Fig. 6 shows the lightinduced difference spectrum of CFRs in the gels at pH 10 over the spectral range from 300 to 800 nm continuous illumination. Maximal depletion by the light of the original pigment was observed at 510 nm. These values were almost the same as the absorption maxima of CFRs at pH 10 shown in Fig. 4 (513 and 511 nm). At 400 nm, an increase of absorbance was observed, implying the formation of an intermediate M of CFR. They all have slower M than GPR. Also, at 600 nm, an increase of absorbance was observed, implying the formation of an intermediated O of CFR. Figure 6, right panel, shows laser-induced absorbance changes of the CFRs at selected wavelength, 400 nm (dash line), 510 nm (solid line), and 600 nm (dash–dot–dot line), which monitor the M intermediate, the depletion of CFR, and the O intermediate, respectively, at the room temperature. The M-decay rate and O-decay rate were estimated by double exponential fitting. The main decay time constants (half time) of the M and O intermediates are presented Table 2. Although GPR exhibits fast M and O decay (<50 ms), three green absorbing CFRs from the Ganges River have slower M and O decay than that of BPR. Discussion We have constructed a cassette with GPR sequence missing helices C to F and inserted the found fragments that contain the helix C to F in it (Fig. 1). The GPR (w/KpnI, NgoMIV) and the BPR/GPR chimera showed characteristic absorption spectra (Fig. 4), indicating no structural perturbation of the retinal binding region by the chimeric protein. Microbial rhodopsins from the environmental samples of fresh water habitats have not been studied yet, although PRs from the oceans are well-known in terms of their sequences, absorption maxima, and photochemical reactions (Beja et al. 2001; Kelemen et al. 2003; Man et al. 2003; ManAharonvich et al. 2004; Sabehi et al. 2005). All chimeric Fig. 5 The left panels show pH dependencies of the relative concen- tration of acid versus alkaline forms of the GPR (w/KpnI and NgoMIV), BPR/GPR, CFR1/GPR, CFR2/GPR, and CFR3/GPR chimeras. Difference spectra were constructed using the spectrum at pH 7.0 as the reference. The pH of purified His-tagged rhodopsins in 50 mM Tris, 150 mM NaCl, and 0.02 % DDM were adjusted with dilute NaOH or HCl. pH titration curves indicate the relative concentration of acid (protonated) form of the pigments. The right panels show proton transporting activities of GPR (w/KpnI and NgoMIV), BPR/GPR chimera, and CFR/GPR chimeras. Illumination (>440 nm) was applied to spheroplasts for 60 after 60 s in the dark period (black boxes), and this cycle was repeated three times. Initial pH values were adjusted to 8.0 Appl Microbiol Biotechnol (2013) 97:819–828 825 826 Appl Microbiol Biotechnol (2013) 97:819–828 Appl Microbiol Biotechnol (2013) 97:819–828 Fig. 6 On the left panel, light-induced difference spectra of GPR (w/ KpnI and NgoMIV), BPR/GPR chimera, and three CFR/GPR chimeras in gel at pH 10 are shown over a spectral range from 300 to 800 nm under the continuous illumination. On the right panel, the O formation and decay, return to the ground state, and the M decay were measured at 600, 510, and 400 nm, respectively freshwater rhodopsin variants in the Ganges River absorb in the green light region and their amino acid sequences are more similar to that of GPR rather than BPR (Beja et al. 2001). Although the chimera proteins are successfully expressed, the pKa values of the Schiff base counterions in CFRs are similar to that of GPR. In case of CFR3, the value is lower than that GPR, suggesting a small structural difference around the chromophore between CFR3 and GPR. In the case of BR/GPR chimera and NpSRII/GPR chimera, the expression of chimeric proteins was not successful due to the structural perturbation of the retinal binding region (data not shown). Also, CFRs must be adapted to its environment, that of freshwater with pH around 7.0, which is lower than in the ocean water (around 8.0). All chimeric freshwater rhodopsins were isolated in this study showed photochemical reactions much slower than that of GPR. Each CFR has several hundred millisecond photocycle like sensory rhodopsins. In general, slow photocycle might be particularly important to distinguish transport rhodopsin from sensory rhodopsin. Though they have slower photocycle kinetics, interestingly, CFRs have proton pumping activity similar to that of GPR. Generally, microbial rhodopsins are widespread all over the world. However, there are some limitations to get full sequence of those from nature. Our findings suggested that this cassette platform exhibits the potential applications to study function of microbial rhodopsins when we can only have partial genome. Also, we will provide many investigators using seven transmembrane ion pumping rhodopsins, the molecular basis of the different spectral properties of partial proteo-opsins that this method will be very easy to speculate on given partial proteo-opsin genes made of short and simple sequences. Acknowledgments This work was supported by the Research Foundation of Korea grants (331-2008-1-C00242 and 2011–0012320) and the second stage of Brain Korea 21 graduate fellowship program for AR Choi, SJ Kim, and BH Jung. We thank Leonid Brown to critical comments on this research. References Atamna-Ismaeel N, Sabehi G, Sharon I, Witzel K-P, Labrenz M, Jurgens K, Barkay T, Stomp M, Huisman J, Beja O (2008) 827 Widespread distribution of proteorhodopsins in freshwater and brackish ecosystems. 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