Light-driven proton-pumping rhodopsins are widely distributed in many microorganisms. They convert sunlight energy into proton gradients that serve as energy source of the cell. Here we report a new functional class of a microbial rhodopsin, a light-driven sodium ion pump. We discover that the marine flavobacterium Krokinobacter eikastus possesses two rhodopsins, the first, KR1, being a prototypical proton pump, while the second, KR2, pumps sodium ions outward. Rhodopsin KR2 can also pump lithium ions, but converts to a proton pump when presented with potassium chloride or salts of larger cations. These data indicate that KR2 is a compatible sodium ion-proton pump, and spectroscopic analysis showed it binds sodium ions in its extracellular domain. These findings suggest that light-driven sodium pumps may be as important in situ as their proton-pumping counterparts.

Perceptual experiences may arise from neuronal activity patterns in mammalian neocortex. We probed mouse neocortex during visual discrimination using a red-shifted channelrhodopsin (ChRmine, discovered through structure-guided genome mining) alongside multiplexed multiphoton-holography (MultiSLM), achieving control of individually specified neurons spanning large cortical volumes with millisecond precision. Stimulating a critical number of stimulus-orientation-selective neurons drove widespread recruitment of functionally related neurons, a process enhanced by (but not requiring) orientation-discrimination task learning. Optogenetic targeting of orientation-selective ensembles elicited correct behavioral discrimination. Cortical layer–specific dynamics were apparent, as emergent neuronal activity asymmetrically propagated from layer 2/3 to layer 5, and smaller layer 5 ensembles were as effective as larger layer 2/3 ensembles in eliciting orientation discrimination behavior. Population dynamics emerging after optogenetic stimulation both correctly predicted behavior and resembled natural internal representations of visual stimuli at cellular resolution over volumes of cortex.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae. Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

Light-activated, ion-pumping rhodopsins are broadly distributed among many different bacteria and archaea inhabiting the photic zone of aquatic environments. Bacterial proton-or sodium-translocating rhodopsins can convert light energy into a chemiosmotic force that can be converted into cellular biochemical energy, and thus represent a widespread alternative form of photoheterotrophy. Here we report that the genome of the marine flavobacterium Nonlabens marinus S1-08 T encodes three different types of rhodopsins: Nonlabens marinus rhodopsin 1 (NM-R1), Nonlabens marinus rhodopsin 2 (NM-R2), and Nonlabens marinus rhodopsin 3 (NM-R3). Our functional analysis demonstrated that NM-R1 and NM-R2 are light-driven outward-translocating H + and Na + pumps, respectively. Functional analyses further revealed that the lightactivated NM-R3 rhodopsin pumps Cl − ions into the cell, representing the first chloride-pumping rhodopsin uncovered in a marine bacterium. Phylogenetic analysis revealed that NM-R3 belongs to a distinct phylogenetic lineage quite distant from archaeal inward Cl − -pumping rhodopsins like halorhodopsin, suggesting that different types of chloride-pumping rhodopsins have evolved independently within marine bacterial lineages. Taken together, our data suggest that similar to haloarchaea, a considerable variety of rhodopsin types with different ion specificities have evolved in marine bacteria, with individual marine strains containing as many as three functionally different rhodopsins.

In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H, Na, and Cl. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl or SO. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl and SO with a subsequent secondary inward H movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl and from 542 to 556 nm for SO, and the affinities of Cl and SO were estimated as 0.112 and 5.81 mM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO) transporter and report its spectroscopic characteristics.

Proteorhodopsin (PR) genes are widely distributed among marine prokaryotes and functions as light-driven proton pump when expressed heterologously in Escherichia coli, suggesting that light energy passing through PR may be substantial in marine environment. However, there are no data on PR proton pump activities in native marine bacteria. Here, we demonstrate light-driven proton pump activity (c. 124 H(+) PR(-1)  min(-1) ) in recently isolated marine Flavobacteria. Among 75 isolates, 38 possessed the PR gene. Illumination of cell suspensions from all eight tested strains in five genera triggered marked pH drops. The action spectrum of proton pump activity closely matched the spectral distribution of the sea surface green light field. Addition of hydroxylamine to a solubilized membrane fraction shifted the spectrum to a form characteristic of PR photobleached into retinal oxime, indicating that PRs in flavobacterial cell membranes transform the photon dose in incident radiation into energy in the form of membrane potential. Our results revealed that PR-mediated proton transport can create the sufficient membrane potential for the ATP synthesis in native flavobacterial cells.

A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29 (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pK values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.