Proteorhodopsin in living color: diversity of spectral properties within living bacterial cells

Proteorhodopsin is a family of over 50 proteins that provide phototrophic capability to marine bacteria by acting as light-powered proton pumps. The potential importance of proteorhodopsin to global ocean ecosystems and the possible applications of proteorhodopsin in optical data storage and optical signal processing have spurred diverse research in this new family of proteins. We show that proteorhodopsin expressed in Escherichia coli is functional and properly inserted in the membrane. At high expression levels, it appears to self-associate. We present a method for determining spectral properties of proteorhodopsin in intact E. coli cells that matches results obtained with detergent-solubilized, purified proteins. Using this method, we observe distinctly different spectra for protonated and deprotonated forms of 21 natural proteorhodopsin proteins in intact E. coli cells. Upon protonation, the wavelength maxima red shifts between 13 and 53 nm. We find that pKa values between 7.1 and 8.5 describe the pH-dependent spectral shift for all of the 21 natural variants of proteorhodopsin. The wavelength maxima of the deprotonated forms of the 21 natural proteorhodopsins cluster in two sequence-related groups: blue proteorhodopsins (B-PR) and green proteorhodopsins (G-PR). The site-directed substitution Leu105Gln in Bac31A8 proteorhodopsin shifts this G-PR's wavelength maximum to a wavelength maximum the same as that of the B-PR Hot75m1 proteorhodopsin. The site-directed substitution Gln107Leu in Hot75m1 proteorhodopsin shifts this B-PR's wavelength maximum to a wavelength maximum as that of Bac31A8 proteorhodopsin.     

Proteorhodopsin photosystem gene clusters exhibit co-evolutionary trends and shared ancestry among diverse marine microbial phyla

Since the recent discovery of retinylidene proteins in marine bacteria (proteorhodopsins), the estimated abundance and diversity of this gene family has expanded rapidly. To explore proteorhodopsin photosystem evolutionary and distributional trends, we identified and compared 16 different proteorhodopsin-containing genome fragments recovered from naturally occurring bacterioplankton populations. In addition to finding several deep-branching proteorhodopsin sequences, proteorhodopsins were found in novel taxonomic contexts, including a betaproteobacterium and a planctomycete. Approximately one-third of the proteorhodopsin-containing genome fragments analysed, as well as a number of recently reported marine bacterial whole genome sequences, contained a linked set of genes required for biosynthesis of the rhodopsin chromophore, retinal. Phylogenetic analyses of the retinal biosynthetic genes suggested their co-evolution and probable coordinated lateral gene transfer into disparate lineages, including Euryarchaeota, Planctomycetales, and three different proteobacterial lineages. The lateral transfer and retention of genes required to assemble a functional proteorhodopsin photosystem appears to be a coordinated and relatively frequent evolutionary event. Strong selection pressure apparently acts to preserve these light-dependent photosystems in diverse marine microbial lineages.     

Diversification and spectral tuning in marine proteorhodopsins

Proteorhodopsins, ubiquitous retinylidene photoactive proton pumps, were recently discovered in the cosmopolitan uncultured SAR86 bacterial group in oceanic surface waters. Two related proteorhodopsin families were found that absorb light with different absorption maxima, 525 nm (green) and 490 nm (blue), and their distribution was shown to be stratified with depth. Using structural modeling comparisons and mutagenesis, we report here on a single amino acid residue at position 105 that functions as a spectral tuning switch and accounts for most of the spectral difference between the two pigment families. Furthermore, looking at natural environments, we found novel proteorhodopsin gene clusters spanning the range of 540-505 nm and containing changes in the same identified key switch residue leading to changes in their absorption maxima. The results suggest a simultaneous diversification of green proteorhodopsin and the new key switch variant pigments. Our observations demonstrate that this single-residue switch mechanism is the major determinant of proteorhodopsin wavelength regulation in natural marine environments.     

Proteorhodopsin genes in giant viruses

Viruses with large genomes encode numerous proteins that do not directly participate in virus biogenesis but rather modify key functional systems of infected cells. We report that a distinct group of giant viruses infecting unicellular eukaryotes that includes Organic Lake Phycodnaviruses and Phaeocystis globosa virus encode predicted proteorhodopsins that have not been previously detected in viruses. Search of metagenomic sequence data shows that putative viral proteorhodopsins are extremely abundant in marine environments. Phylogenetic analysis suggests that giant viruses acquired proteorhodopsins via horizontal gene transfer from proteorhodopsin-encoding protists although the actual donor(s) could not be presently identified. The pattern of conservation of the predicted functionally important amino acid residues suggests that viral proteorhodopsin homologs function as sensory rhodopsins. We hypothesize that viral rhodopsins modulate light-dependent signaling, in particular phototaxis, in infected protists. This article was reviewed by Igor B. Zhulin and Laksminarayan M. Iyer. For the full reviews, see the Reviewers?? reports section.     

New insights into metabolic properties of marine bacteria encoding proteorhodopsins

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.     

Non-ionic detergent assists formation of supercharged nanodiscs and insertion of membrane proteins

Nanodiscs are used to stabilize membrane proteins in a lipid environment and enable investigations of the function and structure of these. Membrane proteins are often only available in small amounts, and thus the stability and ease of use of the nanodiscs are essential. We have recently explored circularizing and supercharging membrane scaffolding proteins (MSPs) for nanodisc formation and found increased temporal stability at elevated temperatures. In the present study, we investigate six different supercharged MSPs and their ability to form nanodiscs: three covalently circularized and the three non-circularized, linear versions. Using standard reconstitution protocols using cholate as the reconstitution detergent, we found that two of the linear constructs formed multiple lipid-protein species, whereas adding n-Dodecyl-B-D-maltoside (DDM) with the cholate in the reconstitution gave rise to single-species nanodisc formation for these MSPs. For all MSPs, the formed nanodiscs were analyzed by small-angle X-ray scattering (SAXS), which showed similar structures for each MSP, respectively, suggesting that the structures of the formed nanodiscs are independent of the initial DDM content, as long as cholate is present. Lastly, we incorporated the membrane protein proteorhodopsin into the supercharged nanodiscs and observed a considerable increase in incorporation yield with the addition of DDM. For the three circularized MSPs, a single major species appeared in the size exclusion chromatography (SEC) chromatogram, suggesting monodisperse nanodiscs with proteorhodopsin incorporated, which is in strong contrast to the samples without DDM showing almost no incorporation and high polydispersity.     

Proteorhodopsin is a light-driven proton pump with variable vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions.The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.     

Actinorhodopsins: proteorhodopsin-like gene sequences found predominantly in non-marine environments

Proteorhodopsins are light-energy-harvesting transmembrane proteins encoded by genes recently discovered in the surface waters of the world's oceans. Metagenomic data from the Global Ocean Sampling expedition (GOS) recovered 2674 proteorhodopsin-related sequences from 51 aquatic samples. Four of these samples were from non-marine environments, specifically, Lake Gatun within the Panama Canal, Delaware Bay and Chesapeake Bay and the Punta Cormorant Lagoon in Ecuador. Rhodopsins related to but phylogenetically distinct from most sequences designated proteorhodopsins were present at all four of these non-marine sites and comprised three different clades that were almost completely absent from marine samples. Phylogenomic analyses of genes adjacent to those encoding these novel rhodopsins suggest affiliation to the Actinobacteria , and hence we propose to name these divergent, non-marine rhodopsins 'actinorhodopsins'. Actinorhodopsins conserve the acidic amino acid residues critical for proton pumping and their genes lack genomic association with those encoding photo-sensory transducer proteins, thus supporting a putative ion pumping function. The ratio of rec A and rad A to rhodopsin genes in the different environment types sampled within the GOS indicates that rhodopsins of one type or another are abundant in microbial communities in freshwater, estuarine and lagoon ecosystems, supporting an important role for these photosystems in all aquatic environments influenced by sunlight.     

Spectroscopic and photochemical characterization of a deep ocean proteorhodopsin

A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20-30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K+-ionophore valinomycin supports that the influx is because of voltage-gated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family.     

Brief Report of a New Highly Divergent Variant of Grapevine leafroll‐associated virus 3 (GLRaV‐3)

The alignment of the complete genomes of genetic variants of Grapevine leafroll‐associated virus 3 (GLRaV‐3) representing phylogenetic groups I, II, III and VI revealed numerous regions with exceptionally high divergence between group I to III and group VI variants. Oligonucleotide primers universal for all the above groups of the virus were designed in conserved short stretches of sequences flanking the divergent regions in the helicase (Hel) and RNA‐dependent RNA polymerase (RdRP) domains of the replicase gene and the divergent copy of the capsid protein (dCP) gene. Cloning and sequencing of the 549‐bp RT‐PCR amplicon of the helicase domain from grapevine cv. Shiraz lead to the detection of a variant of GLRaV‐3, which shared only 69.6–74.1% nt similarity with other variants, including the recently reported, new, highly divergent variant, isolate 139. This was confirmed by the results of the analysis of 517‐bp amplicon of the HSP70 gene of GLRaV‐3 generated in RT‐nested PCR based on degenerate primers for the simultaneous amplification of members of the Closteroviridae family designed by Dovas and Katis (J Virol Methods, 109, 2003, 217). In this genomic region, the variant shares 72.3–78.7% nt similarity with other variants of GLRaV‐3. This previously unreported, new, highly divergent variant was provisionally named GTG10. From the alignment of the HSP70 sequences primers for the specific RT‐nested PCR amplification of the variant GTG10 and members of group VI, and specific simultaneous amplification of variants of groups I, II and III, were designed. The results obtained from brief testing of various grapevines using all these primers suggest a relatively limited presence of GTG10 variant in vineyards.     

ln situ observations on the genus Bathyplotes (Holothuroidea, Echinodermata) in Antarctica and its relevance to taxonomy

Nineteen holothurian specimens were photographed or videotaped in situ on the deeper shelf of the Weddell Sea (Antarctica). These specimens show a different gradient and combinations of characteristics which are found in Bathyplotes rubipunctatus Gutt, 1990. A close relationship to this species is therefore suggested. However, based on external features which are conspicuous in live animals but not visible in preserved material they are classified into five groups. Further investigations will reveal whether these groups are distinct species, or merely variants of B. rubipunctatus .     

Far Eastern mullet Mugil soiuy Basilewsky (Mugilidae, Mugiliformes): the genetic structure of populations and its change under acclimatization

The introduction of Far Eastern mullet (pilengas) in the Azov Sea in the 1970s-1980s has resulted in the formation of a self-reproducing commercial population. We have carried out a comparative population-genetic analysis of the mullet from the native (Primorye, the Sea of Japan basin) and the new (The Azov Sea basin) ranges. Genetic characteristics of three Primorye and three Azov local samples were studied using electrophoretic analysis of 15 enzymes encoded by 21 gene loci. In the Azov mullet, the initial heterozygosity characteristic of the donor population was preserved while the genotype and the allele compositions changed; the changes included a 1.9-fold reduction in the percentage of polymorphic loci and 1.5-fold reduction in the mean number of alleles per locus. The genetic differences between the Azov and the Primorye sample groups were highly significant. In the native range, no genetic differentiation among the mullet samples from different areas was found (Gst = 0.42%), whereas in the Azov Sea basin, the samples from spatially isolated populations (ecological groups) exhibited genetic differences (Gst = 1.38). The genetic divergence of the subpopulations and the excess of heterozygotes at some loci in the Azov mullet suggest selection processes that formed genetically divergent groups associated with the areas of different salinity in the new range. The salinity level is assumed to be the most probable factor of local differentiating selection during fast adaptation and naturalization of the introduced mullet.     

Multiple barriers to recombination between divergent HIV-1 variants revealed by a dual-marker recombination assay

Recombination is a major force for generating human immunodeficiency virus type 1 (HIV-1) diversity and produces numerous recombinants circulating in the human population. We previously established a cell-based system using green fluorescent protein gene (gfp) as a reporter to study the mechanisms of HIV-1 recombination. We now report an improved system capable of detecting recombination using authentic viral sequences. Frameshift mutations were introduced into the gag gene so that parental viruses do not express full-length Gag; however, recombination can generate a progeny virus that expresses a functional Gag. We demonstrate that this Gag reconstitution assay can be used to detect recombination between two group M HIV-1 variants of the same or of different subtypes. Using both gfp and gag assays, we found that, similar to group M viruses, group O viruses also recombine frequently. When recombination between a group M virus and a group O virus was examined, we found three distinct barriers for intergroup recombination. First, similar to recombination within group M viruses, intergroup recombination is affected by the identity of the dimerization initiation signal (DIS); variants with the same DIS recombined at a higher rate than those with different DIS. Second, using the gfp recombination assay, we showed that intergroup recombination occurs much less frequently than intragroup recombination, even though the gfp target sequence is identical in all viruses. Finally, Gag reconstitution between variants from different groups is further reduced compared with green fluorescent protein, indicating that sequence divergence interferes with recombination efficiency in the gag gene. Compared with identical sequences, we estimate that recombination rates are reduced by 3-fold and by 10- to 13-fold when the target regions in gag contain 91% and 72-73% sequence identities, respectively. These results show that there are at least three distinct mechanisms preventing exchange of genetic information between divergent HIV-1 variants from different groups.     

Phylogeographic analysis reveals two periods of divergence in large-scaled redfin Tribolodon hakonensis (Pisces, Cyprinidae)

Analysis of mtDNA variation in one of the amphidromic Far Eastern redfins, Tribolodon hakonensis, revealed the presence of three considerably genetically different mtDNA phylogroups in the individuals from the Russian part of the range. These data suggest the presence of the two periods of divergent evolution in the history of the species examined. Comparison of the haplotype distributions from different phylogroups over the species range revealed geographic localization of only one phylogroup in the population samples from southern continental coastal regions of the Sea of Japan. At the same time, two other phylogroups were found in almost equal ratios in northern continental samples and near the Sakhalin Island. These results suggest that the first stage of the divergent evolution, which occured between Pliocene and Pleistocene, resulted in the formation of genetically isolated form (probably, a species) in the region of the Sea of Japan. The second, later period of divergence, probably associated with the separation of the Sea of Okhotsk from the Pacific Ocean then ended with the integration of earlier genetically separated forms into one species with the common gene pool.     

Far Eastern Mullet Mugil soiuy Basilewsky (Mugilidae,Mugiliformes): The Genetic Structure of Populations and Its Change under Acclimatization

The introduction of Far Eastern mullet (pilengas) in the Azov Sea in the 1970s–1980s has resulted in the formation of a self-reproducing commercial population. We have carried out a comparative population-genetic analysis of the mullet from the native (Primorye, the Sea of Japan basin) and the new (The Azov Sea basin) ranges. Genetic characteristics of three Primorye and three Azov local samples were studied using electrophoretic analysis of 15 enzymes encoded by 21 gene loci. In the Azov mullet, the initial heterozygosity characteristic of the donor population was preserved while the genotype and the allele compositions changed; the changes included a 1.9-fold reduction in the percentage of polymorphic loci and 1.5-fold reduction in the mean number of alleles per locus. The genetic differences between the Azov and the Primorye sample groups were highly significant. In the native range, no genetic differentiation among the mullet samples from different areas was found (G _st = 0.42%), whereas in the Azov Sea basin, the samples from spatially isolated populations (ecological groups) exhibited genetic differences (G _st = 1.38%). The genetic divergence of the subpopulations and the excess of heterozygotes at some loci in the Azov mullet suggest selection processes that formed genetically divergent groups associated with the areas of different salinity in the new range. The salinity level is assumed to be the most probable factor of local differentiating selection during fast adaptation and naturalization of the introduced mullet.     

Disparate Data Sets Resolve Squirrel Monkey (Saimiri) Taxonomy: Implications for Behavioral Ecology and Biomedical Usage

Squirrel monkeys (Saimiri spp.) are the most commonly used neotropical (platyrrhine) monkeys in biomedical research; however, no consensus exists as to the phylogenetic relationships amongst geographic variants or whether these variants represent species or subspecies. Here we report a strongly supported squirrel monkey phylogeny, congruent across multiple data sets, including new field data and the first molecular (mtDNA) cladogram. These data support species-level classification for the three major groups in this study. Approximately the same amount of molecular divergence exists among Saimiri oerstedii, S. sciureus, and S. boliviensis. The S. sciureus/S. oerstedii ancestor diverged from S. boliviensis and shortly thereafter S. sciureus and S. oerstedii diverged. Until now, lack of a robust taxonomy has hindered exploitation of the massive potential of Saimiri for comparative studies. No other primate genus displays such widely divergent, genetically-based social behaviors. Our taxonomy also provides robust support for previous warnings against the widespread use of hybrid squirrel monkeys as research models.     

Wide variation in antibiotic resistance proteins identified by functional metagenomic screening of a soil DNA library

Most genes for antibiotic resistance present in soil microbes remain unexplored because most environmental microbes cannot be cultured. Only recently has the identification of these genes become feasible through the use of culture-independent methods. We screened a soil metagenomic DNA library in an Escherichia coli host for genes that can confer resistance to kanamycin, gentamicin, rifampin, trimethoprim, chloramphenicol, or tetracycline. The screen revealed 41 genes that encode novel protein variants of eight protein families, including aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, and transporters. Several proteins of the same protein family deviate considerably from each other yet confer comparable resistance. For example, five dihydrofolate reductases sharing at most 44% amino acid sequence identity in pairwise comparisons were equivalent in conferring trimethoprim resistance. We identified variants of aminoglycoside acetyltransferases and transporters that differ in the specificity of the drugs for which they confer resistance. We also found wide variation in protein structure. Two forms of rifampin ADP-ribosyltransferases, one twice the size of the other, were similarly effective at conferring rifampin resistance, although the short form was expressed at a much lower level. Functional metagenomic screening provides insight into the large variability in antibiotic resistance protein sequences, revealing divergent variants that preserve protein function.     

Genetic evidence supporting the existence of two distinct species in the genusGasterosteus around Japan

Synopsis The genetic and morphological features ofGasterosteus aculeatus were investigated for 29 populations around Japan. Allozyme analyses recognized two groups (Pacific Ocean group and Japan Sea group) that had distinct characteristic features, and showed high genetic differentiation between them (D = 0.482). The Pacific Ocean group had a wide range, from North America to Japan, along the Pacific coast. The distribution of the Japan Sea group was limited around the Sea of Japan and the Sea of Okhotsk. The distribution of these groups were found to be sympatric on the Pacific coast of Hokkaido Island, Japan. From this area, genetic analyses demonstrated that the sympatric populations of the two groups formed independent breeding stocks, and it is considered that the two groups were reproductively isolated from each other. Additionally, each group had distinctive morphological features of lateral plates and caudal keels in the sympatric area. These results suggested that these two groups of the threespine stickleback comprise different species and that the Japan Sea group is taxonomically distinguishable fromG. aculeatus.     

Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.     

Photochemical characterization of flavobacterial rhodopsin: The importance of the helix E region for heat stability

Although many microbial rhodopsins have been discovered many of organisms in a variety of habitats, little is known about the property and diversity of rhodopsin in flavobacteria. Recent studies discovered that many proteorhodopsin (PR)-like proteins exist in genomes of flavobacteria. Following the isolation of a flavobacterial rhodopsins (FR) from the flavobacteria IMCC1997 from the East Sea of Korea, we characterized its photochemical features. We confirmed that the FR expression is induced by light in the IMCC1997 cell. Upon receiving light energy in vitro, the proton acceptor (D83) and donor (E94) of the FR translocate protons from intracellular to extracellular regions. Compared with proteorhodopsin (PR), the FR from IMCC 1997 cells is very unstable, which may be explained by their primary sequence differences. The ratio of all trans/13-cis retinal conformation does not influence this stability. To measure the stability of FR, we tested heat endurance at 70 °C and found that the heat endurance time of some FR mutants increased. Based upon these results, we found the helix E of this protein to be critical for the unstability of FR.