Many organisms perceive light using rhodopsins (Ernst et al., 2014; Govorunova et al., 2017; Grote et al., 2014; Rozenberg et al., 2021), integral membrane proteins containing retinal chromophores. Rhodopsins are classified into type-1 microbial and type-2 animal rhodopsins, which contain all-trans and 11-cis retinal, respectively. Type-2 animal rhodopsins function as G-protein-coupled receptors, while the molecular functions of type-1 microbial rhodopsins are highly diverse and include light-driven ion pumps, light-gated ion channels, light sensors, and light-activated enzymes. Ion-transporting rhodopsins are used as the main tools to control membrane potential in optogenetics (Deisseroth and Hegemann, 2017). In addition to type-1 and type-2 rhodopsins, a previously unrecognized divergent family of heliorhodopsins (HeRs) was recently discovered using functional metagenomics (Pushkarev et al., 2018). HeRs are distant relatives of type-1 rhodopsins, and their structures and photocycles resemble those of other microbial rhodopsins (Kovalev et al., 2020; Lu et al., 2020; Pushkarev et al., 2018; Shihoya et al., 2019). The most unusual structural feature of HeRs is that their membrane topology is inverted compared to type-1 and -2 rhodopsins (Pushkarev et al., 2018; Shihoya et al., 2019).

Similarly to type-1 rhodopsins, HeRs are encoded in genomes of archaea, bacteria, unicellular eukaryotes, and giant viruses. Physiological functions of HeRs, however, remain unknown. Previous studies did not detect any ion transport activity in HeRs, and based on their slow photocycle, a sensory function was suggested (Pushkarev et al., 2018; Shihoya et al., 2019). Analogously, no ion-transport function is consistent with the crystal structures of the prokaryotic HeRs TaHeR (from a Thermoplasmatales archaeon) (Shihoya et al., 2019) and 48C12 (from an actinobacterium) (Kovalev et al., 2020; Lu et al., 2020), in which the interior of the extracellular half is highly hydrophobic. It should be noted, however, that these structures provide a very limited representation of the family. HeRs are a highly diverse group (Kovalev et al., 2020; Chazan et al., 2022) for which a variety of functions might be expected. In the present study, we report ion transport activity for a viral HeR, which provides support to the idea of functional diversity among HeRs.

The HeRs studied here come from giant double-stranded DNA viruses from the genus Coccolithovirus (Phycodnaviridae) that infect the microalga E. huxleyi (=Gephyrocapsa huxleyi). E. huxleyi is a globally important marine coccolithophore whose massive blooms are observable from satellites and have an impact on Earth’s climate (Thierstein and Young, 2004). E. huxleyi viruses are able to collapse its blooms and thus represent one of the main factors controlling abundance of E. huxleyi in the ocean (Bidle et al., 2007; Vardi et al., 2009). Curiously, coccolithoviruses encode HeRs in their genomes, with some isolates, such as EhV-202, having up to three HeR genes (Figure 1A). One of the HeR genes from EhV-202 (AET42570.1; V2HeR2) has been expressed before and failed to demonstrate any light-dependent ion-transporting activity (Shihoya et al., 2019). Despite this, here we report the detection of photocurrents for a different HeR from EhV-202 as well as several other viral HeRs. We suggest a molecular mechanism for ion transport in these HeRs and discuss their evolution and physiological role.

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Driven by the hypothesis that there might exist a functional diversity among the three HeRs in EhV-202, we first targeted V2HeR3 (AET42421.1), a HeR with only ~30% identity to V2HeR2. We expressed it in a cultured mammalian cell line (ND7/23), visualized its expression by P2A-linked eGFP and a cMyc epitope tag and applied patch-clamp recordings in an attempt to detect photocurrents. Vector expression in ND7/23 cells was confirmed by observing eGFP signal on the cytoplasmic side (green in Figure 1B). Clear membrane fluorescence from anti-cMyc tag was observed only from the C-terminal cMyc, but not from the N-terminal cMyc (magenta in Figure 1B), indicating that the C-terminus faces the extracellular side, as in other HeRs (Pushkarev et al., 2018; Shihoya et al., 2019). Remarkably, the V3HeR-expressing ND7/23 cells yielded consistent photocurrent responses in the patch-clamp experiments (Figure 1C). The photocurrents exhibited a sharp negative transient peak (I₀), which rapidly dropped into a relatively broad peak component (I₁), followed by a steady-state current component (I₂) during the course of illumination. The presence of steady-state currents is an unequivocal indication of ion transport maintained for a long time span (Figure 1—figure supplement 1). Photocurrent amplitude increased with light intensity and demonstrated a single sigmoid curve (Figure 1—figure supplement 2), indicative of photocurrents owing to single-photon events by unphotolyzed molecules. Components I₁ and I₂ exhibited a linear relation to the voltage in the current-voltage (I-V) plot (see Figure 1C), with a reversal potential (E_rev) at +30 and +40 mV, respectively, while I₀ was always negatively directed and exhibits a weak voltage dependency. The passive currents of I₁ and I₂ imply a light-gated channel function (Figure 1C) and the linear I-V relationship suggests that V2HeR3 is a voltage-independent channel.

Next, we focused on the ion selectivity of V2HeR3. The I-V plot under the symmetric ionic conditions on both sides of the membrane without metal cations (pH 7.4) was almost linear with E_rev of about +40 mV (Figure 1C, Figure 1—figure supplement 3). Lowering the extracellular pH from 7.4 to 6.0 resulted in an E_rev shift from +40 to +70 mV (Figure 1D), while a shift from +40 to -6 mV was observed when pH_i was lowered (Figure 1—figure supplement 3). By contrast, replacing the solutions with Na⁺ or K⁺ did not show any significant E_rev shift (Figure 1D), suggesting that V2HeR3 is a light-gated proton channel. There was nevertheless a statistically significant difference in the current amplitude in the presence of K⁺ (Figure 1D). This indicates that the H⁺ transport is somehow enhanced in the presence of K⁺ at the extracellular side.

One might argue that, if V2HeR3 functions as a pure channel, the photocurrent is expected to reverse at 0 mV under symmetric conditions. It should be noted in this respect that channelrhodopsin 2 from Chlamydomonas reinhardtii (ChR2) (Nagel et al., 2003), a standard depolarization tool in optogenetics (Boyden et al., 2005; Deisseroth and Hegemann, 2017; Ishizuka et al., 2006), possesses an outward proton pump activity and is thus a leaky proton pump (Feldbauer et al., 2009). Similarly, at low extracellular pH, the outward proton flux of the proton pump rhodopsin from Gloeobacter violaceus (GR) changes to a passive influx (Vogt et al., 2013). In the case of V2HeR3, significant positive E_rev suggests that V2HeR3 also possesses the activity of a proton pump, although the direction is inward. The E_rev shift upon pH change is smaller than that expected from Nernst potential (Figure 1D). Lowering the extracellular pH from 7.4 to 6.0 (ΔpH = 1.4) is expected to correspond to a shift of 82 mV, yet only a 40–60 mV shift was observed for V2HeR3 (Figure 1D and Figure 1—figure supplement 3B). We thus conclude that the ionic current of V2HeR3 includes an H⁺-pump component which is responsible for the deviation from the Nernst equation.

The current amplitude and the E_rev were also altered depending on the monovalent anions in the solution (Figure 1—figure supplement 4). The inward current (I₁ and I₂) was suppressed in the presence of Cl⁻, Br⁻, and NO₃⁻ in the bath solution (Figure 1—figure supplement 4B right) and the E_rev values shifted accordingly (Figure 1—figure supplement 4B left). On the other hand, no significant change either in the current amplitude or in the E_rev was observed when Asp⁻ in the pipette solution was replaced with Cl⁻ or SO4²⁻ (Figure 1—figure supplement 4C right). Our interpretation of these results is that V2HeR3 contains a binding site for a monovalent anion at the extracellular side, and anion binding affects proton transport. Summarizing these electrophysiological experiments, we conclude that V2HeR3 functions as a proton channel with active inward H⁺ transport. Further support to the idea that V2HeR3 conducts only protons was obtained using an ion transport assay with a pH electrode on V2HeR3 expressed in yeast (Pichia pastoris). Illumination of the V2HeR3-expressing yeast cells increased solution pH, which was abolished by the addition of the proton uncoupler CCCP (Figure 1—figure supplement 5).

Having established the origin of I₁ and I₂, we directed our attention to the initial sharp peak component I₀. The fast inward-directed current I₀ was significantly larger than I₁ and I₂ and the current-voltage relationship of I₀ was dramatically shifted to positive values with respect to the reversal potential (Figure 1C1–V plot, left). Extrapolation of the voltage dependence of I₀ predicted its reversal potential as +290 mV, indicating that I₀ is due to an ion pump component. No significant change was observed when NMG⁺ in the extracellular solution was replaced with Na⁺, or when pH_e was lowered from 7.4 to 6.0 (Figure 1—figure supplement 6A). However, a substantial reduction in the current amplitude was observed when pH_i was lowered to 6.0, but it was not affected by replacement of Na⁺ as the intracellular cation (Figure 1—figure supplement 6A). Further, no significant differences in the I-V plot were observed when comparing Asp⁻, Cl⁻, and SO₄²⁻ (Figure 1—figure supplement 6C and D), suggesting that anions do not contribute to I₀. The photocurrent of the component I_₀ was expanded in Figure 1—figure supplement 7. The component I_₀ reached its negative peak at 0.85 ms, independently from the membrane voltage (Figure 1—figure supplement 7A and B). The peak exhibited exponential relaxation with two time constants (τ_off) (Figure 1—figure supplement 7C). Taken together, these observations could be interpreted as evidence that the inward-directed H⁺ transport and H⁺ exclusion are suppressed by high proton concentrations (pH 6.0) at the cytoplasmic side. In spite of the detailed experiments on the I₀ component above, it is difficult to distinguish ionic current from an intramolecular charge displacement. In fact, several studies proposed that the fast transient peak current in channelrhodopsins and H⁺ pump rhodopsins reflects intramolecular proton transfer from the Schiff base to a proton acceptor residue (Geibel et al., 2001; Sineshchekov et al., 2013). Further experiments would thus be needed to clarify the origin of I₀.

To test the applicability of V2HeR3 for optogenetic manipulation of neuronal activity, we expressed V2HeR3 in cultured cortical neurons. Short light pulses at 505 nm repetitively triggered action potentials in the transfected cells at 1 Hz (Figure 1E and F). Spike probability approached 1.0 at 1 Hz pulse frequency whereas it decreased at 2 and 5 Hz (Figure 1G). The light power required for inducing the action potential was between 2 and 16 mW/mm², in the same range as required for ChR2 (Figure 1—figure supplement 8A and Figure 1—figure supplement 8B; Boyden et al., 2005; Ishizuka et al., 2006). Latency to spike peak equalled 4.7±0.60 ms at 25 mW/mm², but varied with varying light intensity (Figure 1—figure supplement 8C). The neurons remained excitable by current injections at each frequency even after the V2HeR3 expression, confirming that V2HeR3 itself does not harm the neurons (Figure 1H). These results demonstrate that V2HeR3 can be used for optical manipulation of neuronal excitability, although the frequency is limited to up to ca. 1 Hz.

Molecular properties of V2HeR3 were studied for the purified protein heterologously expressed in P. pastoris cells. The purified sample demonstrated an absorption maximum at 500 nm (Figure 2A). HPLC analysis revealed that V2HeR3 contains 64% all-trans retinal in the dark, which was converted to the 13-cis form by light (Figure 2—figure supplement 1). The pK_a of the Schiff base and its counterion were determined to be 14.9 and 4.3, respectively (Figure 2—figure supplement 2), close to those of TaHeR and 48C12 (Pushkarev et al., 2018; Shihoya et al., 2019). The photocycle was found to comprise a series of photointermediates: the primary red-shifted K intermediate, followed by the L intermediate, the deprotonated M intermediate, and the reprotonated O intermediate (Figure 1C). Although the photocycle upon illumination was slow as observed for other HeRs, the M intermediate in V2HeR3 was long-lived and directly returned to the original state (Figure 2B). Formation of the M intermediate possessed μs and ms components, suggesting that the M intermediate is the conducting state of the light-gated proton channel (Figure 2C). We then studied photointermediate states at low temperatures. Figure 2D shows formations of the K and M intermediates at 100 and 230 K, respectively, and their photoequilibria with the unphotolyzed state. FTIR analysis at 77 K (Figure 2—figure supplement 3) revealed a peak pair at 1200 (−)/1193 (+) cm^–1, characteristic of the all-trans to 13-cis photoisomerization (Figure 2E). Similar bands were observed for the M intermediate (Figure 2E and Figure 2—figure supplement 4), although the M intermediate generally lacked positive signals. This may originate from (1) vibration sources other than the retinal, (2) protonated photointermediates, or (3) photointermediates of the 13-cis photocycle. The negative peak at 1657 cm^–1 in Figure 2F is the characteristic vibration of helical amide-I, indicating that a structural perturbation α-helix takes place upon retinal isomerization (in the K intermediate), which is maintained in the M intermediate. Nevertheless, different structural changes in the protein are suggested by the stronger positive peaks at 1628 and 1646 cm^–1 for K and M intermediates, respectively. A peak pair at 1720 (−)/1712 (+) cm^–1 is indicative of a hydrogen-bonding change in a protonated carboxylic acid upon retinal isomerization, while the M intermediate shows peaks at 1734 (+)/1720 (−)/1695 (+) cm^–1 (Figure 2F). TaHeR and 48C12 show no spectral changes in this frequency region (Pushkarev et al., 2018; Shihoya et al., 2019), and thus a protonated carboxylic acid of the C=O stretch at 1720 cm^–1 is unique for V2HeR3.

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The above evidence strongly indicates that the mechanism of ion transport in V2HeR3 is different from other ion-transporting rhodopsins. In order to shed light on this mechanism, we characterized a series of V2HeR3 mutants by the use of site-directed mutagenesis. It is well known that internal carboxylates play an important role in such ion-transporting rhodopsins as ChR2 and bacteriorhodopsin (BR) (Gerwert et al., 2014; Kandori, 2020; Lórenz-Fonfría and Heberle, 2014). V2HeR3 contains 13 carboxylates, among which D2 and E51/E53 are located at the N-terminus and extracellular loop, respectively, excluding the possibility that these residues are involved in the proton transport (Figure 3A). We thus prepared 10 mutants affecting the remaining carboxylate positions in all seven helices, and the photocurrents of the D-to-N or E-to-Q mutants were measured. Figure 3—figure supplements 1 and 2 show the photocurrents and their I-V plots, respectively, measured by expressing each mutant in ND7/23 cells. Absorption spectra of each mutant in ND7/23 and P. pastoris cells were obtained without purification using the hydroxylamine bleaching method (Figure 3—figure supplements 3–5). Among the 10 mutants, those of carboxylates on TM1, TM2, TM4, and TM5 had little effect on the photocurrents, whereas mutations of E105, E191, E205, E215, and E236 on TM3, TM6, and TM7 did have a detectable impact (Figure 3—figure supplement 1). E105Q and E191Q in particular abolished the photocurrent entirely. E105 is the Schiff base counterion, and its neutralization led to loss of visible absorption (Figure 3B bottom). This was not the case for 48C12, as the corresponding mutation exhibited visible absorption upon binding chloride (Singh et al., 2019). In the case of E191Q, visible absorption remained but without photocurrent. The importance of carboxylate at position 191 is demonstrated further by the fact that the E191D mutant retained ion-transporting activity. Nevertheless, the I-V plots suggest that the E191D mutation diminished the proton channel mode converting the protein entirely to an inward proton pump, as indicated by the negative photocurrents. Positions homologous to E191 are I199 and T178 in TaHeR and BR, respectively, which sandwich residues F203 and W182, respectively, with the retinal (Figure 3C). It should be noted that W182 exhibits unique conformational changes important for the proton pumping activity in BR (Nango et al., 2016; Subramaniam and Henderson, 2000; Weinert et al., 2019), and that the highly conserved tryptophan in type-1 microbial rhodopsins is replaced with phenylalanine in most HeRs. Interestingly, the proton-transporting V2HeR3 contains tryptophan at this position, which possibly constitutes the gate for the ion transport together with E191. In Figure 3B, E205Q and E215Q showed positive photocurrent signals at all the membrane voltages tested (−60 to +60 mV), suggesting proton conduction in the opposite direction (outward proton pump). It is thus likely that E205 and E215 are the key residues in defining the direction of proton transport. The current shape and the I-V plot of E236Q are similar to those of the wild type, although a shift of E_rev to the more positive voltage from +30 to +60 mV is observed.

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Despite significant sequence divergence (see Supplementary file 3), HeRs from coccolithoviruses form a monophylum close to HeRs from haptophytes and other algae, including a HeR gene found in E. huxleyi (Figure 4—figure supplement 1). Thus, in an attempt to trace the origins of the ion-transporting activity of V2HeR3, we investigated other viral HeRs, as well as the HeR gene from the host species. In addition to V2HeR3, EhV-202 possesses HeRs from two more distantly related clades, V2HeR1 and V2HeR2, that alone are more widespread among EhV isolates (Figure 4—figure supplement 2). Nevertheless, none of the four tested HeRs from clades V2HeR1 and V2HeR2 exhibited steady-state photocurrents as demonstrated in Figure 4A and Figure 4—figure supplement 3. A similarly negative result was obtained for EhHeR, the HeR from the host alga. We then studied a collection of metagenomic viral HeR genes that were closely related to V2HeR3, as well as the more distantly related HeR from EhV-PS401. Figure 4B and Figure 4—figure supplement 4 clearly show that all of them exhibit ion transport activity similar to that of V2HeR3. The I-V plots of VPS401HeR and VTara8957HeR show that the E_rev is close to 0 mV, indicating that the proton channel mode is dominant (Figure 4B). In contrast, ion-transport properties of VTara5482HeR and VTara4616HeR are very similar to that of V2HeR3 judging from their I-V plots, indicating that the proton pump mode is prominent. The absorption spectra of these proteins obtained by hydroxylamine bleaching are shown in Figure 4—figure supplement 5. A sequence comparison of the HeRs is shown in Figure 4—figure supplement 6, with the key amino acids shown in Figure 4C. E105 of V2HeR3 is the Schiff base counterion, and E215 is conserved among HeRs. Among the other carboxylate residues, E205 and E236 are not fully conserved among ion-transporting HeRs, while E191 appears to be their hallmark. Interestingly, although relatively uncommon among eukaryotic HeRs, EhHeR that did not demonstrate ion transport, as well as the more distantly related McHeR that was tested before (Shihoya et al., 2019), also contain the conserved glutamate E191 (see Figure 4C and Figure 4—figure supplement 1). This indicates that E191 might be essential but not sufficient for ion transport, and that other residues, such as W195 and E/Q205, are required as well.

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Figure 4D outlines the proposed mechanism of proton channeling and pumping among the viral HeRs. Inward proton pumps were recently found in nature (Harris et al., 2020; Inoue et al., 2020; Inoue et al., 2016; Shevchenko et al., 2017), but the mechanism should be compared with that of outward proton pumps like BR, as the membrane topology in HeRS is opposite to that of type-1 and -2 rhodopsins (Figure 1B). Crystal structures of TaHeR and 48C12 showed water-containing hydrogen-bonding networks between the retinal Schiff base and cytoplasmic aqueous phase. While the interior of the extracellular half is entirely hydrophobic in TaHeR (Shihoya et al., 2019) and 48C12 (Kovalev et al., 2020; Lu et al., 2020), the proton-transporting HeRs reported here contain polar residues in that region. In particular, E191 appears to be the key residue for the function, presumably constituting the channel gate together with W195. Although E191 is a prerequisite for ion transport, it does not mean that this residue is negatively charged. Rather, E191 is protonated in the unphotolyzed state, and its deprotonation occurs upon opening of the gate. This residue is analogous to D96 in TM3 of BR and E90 in TM2 of ChR2, both of which are protonated in the dark state with the transient deprotonation necessary for light-driven proton pumping and light-gated cation channeling activities, respectively (Braiman et al., 1988; Gerwert et al., 1989; Kuhne et al., 2019; Radu et al., 2009). Similarly, in the cation channelrhodopsin 2 from Guillardia theta (GtCCR2) that shares with BR the DTD motif in TM3, channel opening requires deprotonation of the D96 homolog (Sineshchekov et al., 2017). Mutation of E205 or E215 in V2HeR3 led to conversion of proton pumping direction which suggests that E205 and E215 constitute the proton accepting group (PAG) upon formation of the M intermediate. These residues correspond to Q213 and E227 in TaHeR, and Q216 and E230 in 48C12, which constitute a water-containing hydrogen-bonding network (Kovalev et al., 2020; Lu et al., 2020; Shihoya et al., 2019). As HeRs contain a single counterion of the Schiff base, it cannot be the proton acceptor upon deprotonation of the Schiff base. Instead, in the case of V2HeR3, the E205/E215 region appears to serve as PAG that receives the proton from the Schiff base. Interestingly, when the PAG mechanism is disrupted by mutation, the protein is converted to an outward proton pump. We found that monovalent anions affect E_rev, but only at the extracellular side (Figure 1—figure supplement 4), suggesting anion binding to V2HeR3 (Figure 4D).

The discovery of proton-transporting HeRs provides various insights regarding evolution, physiology, mechanism of action, and application of this rhodopsin family. Phylogenetic analysis strongly indicates that coccolithoviruses acquired their HeR genes from their past algal hosts (see Figure 4—figure supplement 1). The split into proton-transporting and non-transporting types took place early in the evolution of the viral HeRs, while their distribution among coccolithoviruses suggests that the last common ancestor of all known isolates might have already possessed genes of both types (Figure 4—figure supplement 2). The proton-transporting HeR was then secondarily lost in the lineage of EhV-201. Proton-transporting HeRs are thus an innovation that appeared after the viruses acquired HeRs from algae, but the exact benefit that this function provides to the viruses is unclear. It was reported that light is required for viral adsorption to E. huxleyi cells (Thamatrakoln et al., 2019). If V2HeR3 is expressed in E. huxleyi cell membranes, light might depolarize the host cell membranes by this protein. It can be thus speculated that depolarization helps the virus to overcome cell defense or prevents superinfection. As E. huxleyi blooms significantly affect the marine environment and climate, the influence of light is intriguing. The viral HeRs are the first rhodopsin transporters to be characterized from the family Phycodnaviridae, paralleled by the type-1 rhodopsin channels in the Mimiviridae (Rozenberg et al., 2020; Zabelskii et al., 2020).

Type-1 microbial rhodopsins’ functions are diverse, while we now know that some HeRs participate in ion-transport such as proton channeling and pumping. The functional conversion of V2HeR3 into an outward proton pump may suggest the presence of such HeR proteins in nature. Thus, HeR’s functions are likely to be diverse similarly to type-1 rhodopsins. Finally, type-1 ion-transporting rhodopsins have been used as the main tools in optogenetics (Boyden et al., 2005; Deisseroth and Hegemann, 2017; Ishizuka et al., 2006), and ion-transporting HeRs could be potential optogenetic tools as well.

All data needed to evaluate the conclusions are present in the paper. The datasets of the current study are available in the Dryad repository (https://doi.org/10.5061/dryad.31zcrjdpb).

1. 1. Kandori H

   (2022) Dryad Digital Repository

   Proton-transporting heliorhodopsins from marine giant viruses.

   https://doi.org/10.5061/dryad.31zcrjdpb

1. 1. Altschul SF
   2. Gish W
   3. Miller W
   4. Myers EW
   5. Lipman DJ

   (1990) Basic local alignment search tool

   Journal of Molecular Biology 215:403–410.

   https://doi.org/10.1016/S0022-2836(05)80360-2

   • PubMed
   • Google Scholar
2. 1. Bidle KD
   2. Haramaty L
   3. Barcelos E Ramos J
   4. Falkowski P

   (2007) Viral activation and recruitment of metacaspases in the unicellular coccolithophore, emiliania huxleyi

   PNAS 104:6049–6054.

   https://doi.org/10.1073/pnas.0701240104

   • PubMed
   • Google Scholar
3. 1. Boyden ES
   2. Zhang F
   3. Bamberg E
   4. Nagel G
   5. Deisseroth K

   (2005) Millisecond-timescale, genetically targeted optical control of neural activity

   Nature Neuroscience 8:1263–1268.

   https://doi.org/10.1038/nn1525

   • PubMed
   • Google Scholar
4. 1. Braiman MS
   2. Mogi T
   3. Marti T
   4. Stern LJ
   5. Khorana HG
   6. Rothschild KJ

   (1988) Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212

   Biochemistry 27:8516–8520.

   https://doi.org/10.1021/bi00423a002

   • PubMed
   • Google Scholar
5. 1. Capella-Gutiérrez S
   2. Silla-Martínez JM
   3. Gabaldón T

   (2009) TrimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses

   Bioinformatics 25:1972–1973.

   https://doi.org/10.1093/bioinformatics/btp348

   • PubMed
   • Google Scholar
6. 1. Chazan A
   2. Rozenberg A
   3. Mannen K
   4. Nagata T
   5. Tahan R
   6. Yaish S
   7. Larom S
   8. Inoue K
   9. Béjà O
   10. Pushkarev A

   (2022) Diverse heliorhodopsins detected via functional metagenomics in freshwater actinobacteria, chloroflexi and archaea

   Environmental Microbiology 24:110–121.

   https://doi.org/10.1111/1462-2920.15890

   • PubMed
   • Google Scholar
7. 1. Chen K
   2. Durand D
   3. Farach-Colton M

   (2000) NOTUNG: a program for dating gene duplications and optimizing gene family trees

   Journal of Computational Biology 7:429–447.

   https://doi.org/10.1089/106652700750050871

   • PubMed
   • Google Scholar
8. 1. Deisseroth K
   2. Hegemann P

   (2017) The form and function of channelrhodopsin

   Science 357:eaan5544.

   https://doi.org/10.1126/science.aan5544

   • PubMed
   • Google Scholar
9. 1. Eddy SR

   (1996) Hidden markov models

   Current Opinion in Structural Biology 6:361–365.

   https://doi.org/10.1016/s0959-440x(96)80056-x

   • PubMed
   • Google Scholar
10. 1. Edgar RC

    (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput

    Nucleic Acids Research 32:1792–1797.

    https://doi.org/10.1093/nar/gkh340

    • PubMed
    • Google Scholar
11. 1. Ernst OP
    2. Lodowski DT
    3. Elstner M
    4. Hegemann P
    5. Brown LS
    6. Kandori H

    (2014) Microbial and animal rhodopsins: structures, functions, and molecular mechanisms

    Chemical Reviews 114:126–163.

    https://doi.org/10.1021/cr4003769

    • PubMed
    • Google Scholar
12. 1. Feldbauer K
    2. Zimmermann D
    3. Pintschovius V
    4. Spitz J
    5. Bamann C
    6. Bamberg E

    (2009) Channelrhodopsin-2 is a leaky proton pump

    PNAS 106:12317–12322.

    https://doi.org/10.1073/pnas.0905852106

    • PubMed
    • Google Scholar
13. 1. Geibel S
    2. Friedrich T
    3. Ormos P
    4. Wood PG
    5. Nagel G
    6. Bamberg E

    (2001) The voltage-dependent proton pumping in bacteriorhodopsin is characterized by optoelectric behavior

    Biophysical Journal 81:2059–2068.

    https://doi.org/10.1016/S0006-3495(01)75855-9

    • PubMed
    • Google Scholar
14. 1. Gerwert K
    2. Hess B
    3. Soppa J
    4. Oesterhelt D

    (1989) Role of aspartate-96 in proton translocation by bacteriorhodopsin

    PNAS 86:4943–4947.

    https://doi.org/10.1073/pnas.86.13.4943

    • PubMed
    • Google Scholar
15. 1. Gerwert K
    2. Freier E
    3. Wolf S

    (2014) The role of protein-bound water molecules in microbial rhodopsins

    Biochimica et Biophysica Acta 1837:606–613.

    https://doi.org/10.1016/j.bbabio.2013.09.006

    • PubMed
    • Google Scholar
16. 1. Govorunova EG
    2. Sineshchekov OA
    3. Li H
    4. Spudich JL

    (2017) Microbial rhodopsins: diversity, mechanisms, and optogenetic applications

    Annual Review of Biochemistry 86:845–872.

    https://doi.org/10.1146/annurev-biochem-101910-144233

    • PubMed
    • Google Scholar
17. 1. Grote M
    2. Engelhard M
    3. Hegemann P

    (2014) Of ion pumps, sensors and channels - perspectives on microbial rhodopsins between science and history

    Biochimica et Biophysica Acta 1837:533–545.

    https://doi.org/10.1016/j.bbabio.2013.08.006

    • PubMed
    • Google Scholar
18. 1. Haas BJ
    2. Papanicolaou A
    3. Yassour M
    4. Grabherr M
    5. Blood PD
    6. Bowden J
    7. Couger MB
    8. Eccles D
    9. Li B
    10. Lieber M
    11. MacManes MD
    12. Ott M
    13. Orvis J
    14. Pochet N
    15. Strozzi F
    16. Weeks N
    17. Westerman R
    18. William T
    19. Dewey CN
    20. Henschel R
    21. LeDuc RD
    22. Friedman N
    23. Regev A

    (2013) De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis

    Nature Protocols 8:1494–1512.

    https://doi.org/10.1038/nprot.2013.084

    • PubMed
    • Google Scholar
19. 1. Harris A
    2. Lazaratos M
    3. Siemers M
    4. Watt E
    5. Hoang A
    6. Tomida S
    7. Schubert L
    8. Saita M
    9. Heberle J
    10. Furutani Y
    11. Kandori H
    12. Bondar AN
    13. Brown LS

    (2020) Mechanism of inward proton transport in an antarctic microbial rhodopsin

    The Journal of Physical Chemistry. B 124:4851–4872.

    https://doi.org/10.1021/acs.jpcb.0c02767

    • PubMed
    • Google Scholar
20. 1. Hososhima S
    2. Kandori H
    3. Tsunoda SP

    (2021) Ion transport activity and optogenetics capability of light-driven na+-pump KR2

    PLOS ONE 16:e0256728.

    https://doi.org/10.1371/journal.pone.0256728

    • PubMed
    • Google Scholar
21. 1. Inoue K
    2. Ono H
    3. Abe-Yoshizumi R
    4. Yoshizawa S
    5. Ito H
    6. Kogure K
    7. Kandori H

    (2013) A light-driven sodium ion pump in marine bacteria

    Nature Communications 4:1678.

    https://doi.org/10.1038/ncomms2689

    • PubMed
    • Google Scholar
22. 1. Inoue K
    2. Ito S
    3. Kato Y
    4. Nomura Y
    5. Shibata M
    6. Uchihashi T
    7. Tsunoda SP
    8. Kandori H

    (2016) A natural light-driven inward proton pump

    Nature Communications 7:13415.

    https://doi.org/10.1038/ncomms13415

    • PubMed
    • Google Scholar
23. 1. Inoue K
    2. Tsunoda SP
    3. Singh M
    4. Tomida S
    5. Hososhima S
    6. Konno M
    7. Nakamura R
    8. Watanabe H
    9. Bulzu PA
    10. Banciu HL
    11. Andrei AŞ
    12. Uchihashi T
    13. Ghai R
    14. Béjà O
    15. Kandori H

    (2020) Schizorhodopsins: A family of rhodopsins from asgard archaea that function as light-driven inward H⁺ pumps

    Science Advances 6:eaaz2441.

    https://doi.org/10.1126/sciadv.aaz2441

    • PubMed
    • Google Scholar
24. 1. Ishizuka T
    2. Kakuda M
    3. Araki R
    4. Yawo H

    (2006) Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels

    Neuroscience Research 54:85–94.

    https://doi.org/10.1016/j.neures.2005.10.009

    • PubMed
    • Google Scholar
25. 1. Johnson LK
    2. Alexander H
    3. Brown CT

    (2019) Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes

    GigaScience 8:giy158.

    https://doi.org/10.1093/gigascience/giy158

    • PubMed
    • Google Scholar
26. 1. Käll L
    2. Krogh A
    3. Sonnhammer ELL

    (2005) An HMM posterior decoder for sequence feature prediction that includes homology information

    BBioinformatics 21 Suppl 1:i251–i257.

    https://doi.org/10.1093/bioinformatics/bti1014

    • PubMed
    • Google Scholar
27. 1. Kandori H

    (2020) Structure/function study of photoreceptive proteins by FTIR spectroscopy

    Bulletin of the Chemical Society of Japan 93:904–926.

    https://doi.org/10.1246/bcsj.20200109

    • Google Scholar
28. 1. Katoh K
    2. Misawa K
    3. Kuma K
    4. Miyata T

    (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast fourier transform

    Nucleic Acids Research 30:3059–3066.

    https://doi.org/10.1093/nar/gkf436

    • PubMed
    • Google Scholar
29. 1. Kawanabe A
    2. Furutani Y
    3. Jung KH
    4. Kandori H

    (2006) FTIR study of the photoisomerization processes in the 13-cis and all-trans forms of anabaena sensory rhodopsin at 77 K

    Biochemistry 45:4362–4370.

    https://doi.org/10.1021/bi052324b

    • PubMed
    • Google Scholar
30. 1. Kawanabe A
    2. Furutani Y
    3. Jung KH
    4. Kandori H

    (2007) Photochromism of anabaena sensory rhodopsin

    Journal of the American Chemical Society 129:8644–8649.

    https://doi.org/10.1021/ja072085a

    • PubMed
    • Google Scholar
31. 1. Keeling PJ
    2. Burki F
    3. Wilcox HM
    4. Allam B
    5. Allen EE
    6. Amaral-Zettler LA
    7. Armbrust EV
    8. Archibald JM
    9. Bharti AK
    10. Bell CJ
    11. Beszteri B
    12. Bidle KD
    13. Cameron CT
    14. Campbell L
    15. Caron DA
    16. Cattolico RA
    17. Collier JL
    18. Coyne K
    19. Davy SK
    20. Deschamps P
    21. Dyhrman ST
    22. Edvardsen B
    23. Gates RD
    24. Gobler CJ
    25. Greenwood SJ
    26. Guida SM
    27. Jacobi JL
    28. Jakobsen KS
    29. James ER
    30. Jenkins B
    31. John U
    32. Johnson MD
    33. Juhl AR
    34. Kamp A
    35. Katz LA
    36. Kiene R
    37. Kudryavtsev A
    38. Leander BS
    39. Lin S
    40. Lovejoy C
    41. Lynn D
    42. Marchetti A
    43. McManus G
    44. Nedelcu AM
    45. Menden-Deuer S
    46. Miceli C
    47. Mock T
    48. Montresor M
    49. Moran MA
    50. Murray S
    51. Nadathur G
    52. Nagai S
    53. Ngam PB
    54. Palenik B
    55. Pawlowski J
    56. Petroni G
    57. Piganeau G
    58. Posewitz MC
    59. Rengefors K
    60. Romano G
    61. Rumpho ME
    62. Rynearson T
    63. Schilling KB
    64. Schroeder DC
    65. Simpson AGB
    66. Slamovits CH
    67. Smith DR
    68. Smith GJ
    69. Smith SR
    70. Sosik HM
    71. Stief P
    72. Theriot E
    73. Twary SN
    74. Umale PE
    75. Vaulot D
    76. Wawrik B
    77. Wheeler GL
    78. Wilson WH
    79. Xu Y
    80. Zingone A
    81. Worden AZ

    (2014) The marine microbial eukaryote transcriptome sequencing project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing

    PLOS Biology 12:e1001889.

    https://doi.org/10.1371/journal.pbio.1001889

    • PubMed
    • Google Scholar
32. 1. Kovalev K
    2. Volkov D
    3. Astashkin R
    4. Alekseev A
    5. Gushchin I
    6. Haro-Moreno JM
    7. Chizhov I
    8. Siletsky S
    9. Mamedov M
    10. Rogachev A
    11. Balandin T
    12. Borshchevskiy V
    13. Popov A
    14. Bourenkov G
    15. Bamberg E
    16. Rodriguez-Valera F
    17. Büldt G
    18. Gordeliy V

    (2020) High-resolution structural insights into the heliorhodopsin family

    PNAS 117:4131–4141.

    https://doi.org/10.1073/pnas.1915888117

    • PubMed
    • Google Scholar
33. 1. Kuhne J
    2. Vierock J
    3. Tennigkeit SA
    4. Dreier MA
    5. Wietek J
    6. Petersen D
    7. Gavriljuk K
    8. El-Mashtoly SF
    9. Hegemann P
    10. Gerwert K

    (2019) Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2

    PNAS 116:9380–9389.

    https://doi.org/10.1073/pnas.1818707116

    • PubMed
    • Google Scholar
34. 1. Kumar S
    2. Stecher G
    3. Li M
    4. Knyaz C
    5. Tamura K

    (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms

    Molecular Biology and Evolution 35:1547–1549.

    https://doi.org/10.1093/molbev/msy096

    • PubMed
    • Google Scholar
35. 1. Lechner M
    2. Findeiss S
    3. Steiner L
    4. Marz M
    5. Stadler PF
    6. Prohaska SJ

    (2011) Proteinortho: detection of (co-)orthologs in large-scale analysis

    BMC Bioinformatics 12:124.

    https://doi.org/10.1186/1471-2105-12-124

    • PubMed
    • Google Scholar
36. 1. Leebens-Mack JH

    (2019) One thousand plant transcriptomes and the phylogenomics of green plants

    Nature 574:679–685.

    https://doi.org/10.1038/s41586-019-1693-2

    • PubMed
    • Google Scholar
37. 1. Li W
    2. Godzik A

    (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences

    Bioinformatics 22:1658–1659.

    https://doi.org/10.1093/bioinformatics/btl158

    • PubMed
    • Google Scholar
38. 1. Liew YJ
    2. Aranda M
    3. Voolstra CR

    (2016) Reefgenomics.org - a repository for marine genomics data

    Database 2016:baw152.

    https://doi.org/10.1093/database/baw152

    • PubMed
    • Google Scholar
39. 1. Lomsadze A
    2. Ter-Hovhannisyan V
    3. Chernoff YO
    4. Borodovsky M

    (2005) Gene identification in novel eukaryotic genomes by self-training algorithm

    Nucleic Acids Research 33:6494–6506.

    https://doi.org/10.1093/nar/gki937

    • PubMed
    • Google Scholar
40. 1. Lórenz-Fonfría VA
    2. Heberle J

    (2014) Channelrhodopsin unchained: structure and mechanism of a light-gated cation channel

    Biochimica et Biophysica Acta 1837:626–642.

    https://doi.org/10.1016/j.bbabio.2013.10.014

    • PubMed
    • Google Scholar
41. 1. Lu Y
    2. Zhou XE
    3. Gao X
    4. Wang N
    5. Xia R
    6. Xu Z
    7. Leng Y
    8. Shi Y
    9. Wang G
    10. Melcher K
    11. Xu HE
    12. He Y

    (2020) Crystal structure of heliorhodopsin 48C12

    Cell Research 30:88–90.

    https://doi.org/10.1038/s41422-019-0266-0

    • PubMed
    • Google Scholar
42. 1. Minh BQ
    2. Schmidt HA
    3. Chernomor O
    4. Schrempf D
    5. Woodhams MD
    6. von Haeseler A
    7. Lanfear R

    (2020) IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era

    Molecular Biology and Evolution 37:1530–1534.

    https://doi.org/10.1093/molbev/msaa015

    • PubMed
    • Google Scholar
43. 1. Nagel G
    2. Szellas T
    3. Huhn W
    4. Kateriya S
    5. Adeishvili N
    6. Berthold P
    7. Ollig D
    8. Hegemann P
    9. Bamberg E

    (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel

    PNAS 100:13940–13945.

    https://doi.org/10.1073/pnas.1936192100

    • PubMed
    • Google Scholar
44. 1. Nango E
    2. Royant A
    3. Kubo M
    4. Nakane T
    5. Wickstrand C
    6. Kimura T
    7. Tanaka T
    8. Tono K
    9. Song C
    10. Tanaka R
    11. Arima T
    12. Yamashita A
    13. Kobayashi J
    14. Hosaka T
    15. Mizohata E
    16. Nogly P
    17. Sugahara M
    18. Nam D
    19. Nomura T
    20. Shimamura T
    21. Im D
    22. Fujiwara T
    23. Yamanaka Y
    24. Jeon B
    25. Nishizawa T
    26. Oda K
    27. Fukuda M
    28. Andersson R
    29. Båth P
    30. Dods R
    31. Davidsson J
    32. Matsuoka S
    33. Kawatake S
    34. Murata M
    35. Nureki O
    36. Owada S
    37. Kameshima T
    38. Hatsui T
    39. Joti Y
    40. Schertler G
    41. Yabashi M
    42. Bondar AN
    43. Standfuss J
    44. Neutze R
    45. Iwata S

    (2016) A three-dimensional movie of structural changes in bacteriorhodopsin

    Science 354:1552–1557.

    https://doi.org/10.1126/science.aah3497

    • PubMed
    • Google Scholar
45. 1. Nissimov JI
    2. Worthy CA
    3. Rooks P
    4. Napier JA
    5. Kimmance SA
    6. Henn MR
    7. Ogata H
    8. Allen MJ

    (2012a) Draft genome sequence of four coccolithoviruses: emiliania huxleyi virus ehv-88, ehv-201, ehv-207, and ehv-208

    Journal of Virology 86:2896–2897.

    https://doi.org/10.1128/JVI.07046-11

    • PubMed
    • Google Scholar
46. 1. Nissimov JI
    2. Worthy CA
    3. Rooks P
    4. Napier JA
    5. Kimmance SA
    6. Henn MR
    7. Ogata H
    8. Allen MJ

    (2012b) Draft genome sequence of the coccolithovirus emiliania huxleyi virus 202

    Journal of Virology 86:2380–2381.

    https://doi.org/10.1128/JVI.06863-11

    • PubMed
    • Google Scholar
47. 1. Philosof A
    2. Yutin N
    3. Flores-Uribe J
    4. Sharon I
    5. Koonin EV
    6. Béjà O

    (2017) Novel abundant oceanic viruses of uncultured marine group II euryarchaeota

    Current Biology 27:1362–1368.

    https://doi.org/10.1016/j.cub.2017.03.052

    • PubMed
    • Google Scholar
48. 1. Pushkarev A
    2. Inoue K
    3. Larom S
    4. Flores-Uribe J
    5. Singh M
    6. Konno M
    7. Tomida S
    8. Ito S
    9. Nakamura R
    10. Tsunoda SP
    11. Philosof A
    12. Sharon I
    13. Yutin N
    14. Koonin EV
    15. Kandori H
    16. Béjà O

    (2018) A distinct abundant group of microbial rhodopsins discovered using functional metagenomics

    Nature 558:595–599.

    https://doi.org/10.1038/s41586-018-0225-9

    • PubMed
    • Google Scholar
49. 1. Radu I
    2. Bamann C
    3. Nack M
    4. Nagel G
    5. Bamberg E
    6. Heberle J

    (2009) Conformational changes of channelrhodopsin-2

    Journal of the American Chemical Society 131:7313–7319.

    https://doi.org/10.1021/ja8084274

    • PubMed
    • Google Scholar
50. 1. Rozenberg A
    2. Oppermann J
    3. Wietek J
    4. Fernandez Lahore RG
    5. Sandaa RA
    6. Bratbak G
    7. Hegemann P
    8. Béjà O

    (2020) Lateral gene transfer of anion-conducting channelrhodopsins between green algae and giant viruses

    Current Biology 30:4910–4920.

    https://doi.org/10.1016/j.cub.2020.09.056

    • PubMed
    • Google Scholar
51. 1. Rozenberg A
    2. Inoue K
    3. Kandori H
    4. Béjà O

    (2021) Microbial rhodopsins: the last two decades

    Annual Review of Microbiology 75:427–447.

    https://doi.org/10.1146/annurev-micro-031721-020452

    • PubMed
    • Google Scholar
52. Software

    1. Rozenberg A

    (2022) Viral heliorhodopsins pipeline, version swh:1:rev:0a2cc9a502c5d0d9dbc6dbcac962d89c3a5a254d

    Software Heritage.

    https://archive.softwareheritage.org/swh:1:dir:aaa9afbd4479761d9f48245eaab08247a67e1b47;origin=https://github.com/BejaLab/ViralHeRs;visit=swh:1:snp:d44a4e804066a14e49243c1bdd6a1605242d411c;anchor=swh:1:rev:0a2cc9a502c5d0d9dbc6dbcac962d89c3a5a254d
53. 1. Saitou N
    2. Nei M

    (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees

    Molecular Biology and Evolution 4:406–425.

    https://doi.org/10.1093/oxfordjournals.molbev.a040454

    • PubMed
    • Google Scholar
54. 1. Schindelin J
    2. Arganda-Carreras I
    3. Frise E
    4. Kaynig V
    5. Longair M
    6. Pietzsch T
    7. Preibisch S
    8. Rueden C
    9. Saalfeld S
    10. Schmid B
    11. Tinevez JY
    12. White DJ
    13. Hartenstein V
    14. Eliceiri K
    15. Tomancak P
    16. Cardona A

    (2012) Fiji: an open-source platform for biological-image analysis

    Nature Methods 9:676–682.

    https://doi.org/10.1038/nmeth.2019

    • PubMed
    • Google Scholar
55. 1. Shevchenko V
    2. Mager T
    3. Kovalev K
    4. Polovinkin V
    5. Alekseev A
    6. Juettner J
    7. Chizhov I
    8. Bamann C
    9. Vavourakis C
    10. Ghai R
    11. Gushchin I
    12. Borshchevskiy V
    13. Rogachev A
    14. Melnikov I
    15. Popov A
    16. Balandin T
    17. Rodriguez-Valera F
    18. Manstein DJ
    19. Bueldt G
    20. Bamberg E
    21. Gordeliy V

    (2017) Inward h ⁺ pump xenorhodopsin: mechanism and alternative optogenetic approach

    Science Advances 3:e03187.

    https://doi.org/10.1126/sciadv.1603187

    • Google Scholar
56. 1. Shihoya W
    2. Inoue K
    3. Singh M
    4. Konno M
    5. Hososhima S
    6. Yamashita K
    7. Ikeda K
    8. Higuchi A
    9. Izume T
    10. Okazaki S
    11. Hashimoto M
    12. Mizutori R
    13. Tomida S
    14. Yamauchi Y
    15. Abe-Yoshizumi R
    16. Katayama K
    17. Tsunoda SP
    18. Shibata M
    19. Furutani Y
    20. Pushkarev A
    21. Béjà O
    22. Uchihashi T
    23. Kandori H
    24. Nureki O

    (2019) Crystal structure of heliorhodopsin

    Nature 574:132–136.

    https://doi.org/10.1038/s41586-019-1604-6

    • PubMed
    • Google Scholar
57. 1. Sineshchekov OA
    2. Govorunova EG
    3. Wang J
    4. Li H
    5. Spudich JL

    (2013) Intramolecular proton transfer in channelrhodopsins

    Biophysical Journal 104:807–817.

    https://doi.org/10.1016/j.bpj.2013.01.002

    • PubMed
    • Google Scholar
58. 1. Sineshchekov OA
    2. Govorunova EG
    3. Li H
    4. Spudich JL

    (2017) Bacteriorhodopsin-like channelrhodopsins: alternative mechanism for control of cation conductance

    PNAS 114:E9512–E9519.

    https://doi.org/10.1073/pnas.1710702114

    • PubMed
    • Google Scholar
59. 1. Singh M
    2. Katayama K
    3. Béjà O
    4. Kandori H

    (2019) Anion binding to mutants of the schiff base counterion in heliorhodopsin 48C12

    Physical Chemistry Chemical Physics 21:23663–23671.

    https://doi.org/10.1039/c9cp04102h

    • PubMed
    • Google Scholar
60. 1. Subramaniam S
    2. Henderson R

    (2000) Molecular mechanism of vectorial proton translocation by bacteriorhodopsin

    Nature 406:653–657.

    https://doi.org/10.1038/35020614

    • PubMed
    • Google Scholar
61. 1. Sunagawa S
    2. Coelho LP
    3. Chaffron S
    4. Kultima JR
    5. Labadie K
    6. Salazar G
    7. Djahanschiri B
    8. Zeller G
    9. Mende DR
    10. Alberti A
    11. Cornejo-Castillo FM
    12. Costea PI
    13. Cruaud C
    14. d’Ovidio F
    15. Engelen S
    16. Ferrera I
    17. Gasol JM
    18. Guidi L
    19. Hildebrand F
    20. Kokoszka F
    21. Lepoivre C
    22. Lima-Mendez G
    23. Poulain J
    24. Poulos BT
    25. Royo-Llonch M
    26. Sarmento H
    27. Vieira-Silva S
    28. Dimier C
    29. Picheral M
    30. Searson S
    31. Kandels-Lewis S
    32. Bowler C
    33. de Vargas C
    34. Gorsky G
    35. Grimsley N
    36. Hingamp P
    37. Iudicone D
    38. Jaillon O
    39. Not F
    40. Ogata H
    41. Pesant S
    42. Speich S
    43. Stemmann L
    44. Sullivan MB
    45. Weissenbach J
    46. Wincker P
    47. Karsenti E
    48. Raes J
    49. Acinas SG
    50. Bork P
    51. Tara Oceans coordinators

    (2015) Ocean plankton. Structure and function of the global ocean microbiome

    Science 348:1261359.

    https://doi.org/10.1126/science.1261359

    • PubMed
    • Google Scholar
62. 1. Thamatrakoln K
    2. Talmy D
    3. Haramaty L
    4. Maniscalco C
    5. Latham JR
    6. Knowles B
    7. Natale F
    8. Coolen MJL
    9. Follows MJ
    10. Bidle KD

    (2019) Light regulation of coccolithophore host-virus interactions

    The New Phytologist 221:1289–1302.

    https://doi.org/10.1111/nph.15459

    • PubMed
    • Google Scholar
63. Book

    1. Thierstein HR
    2. Young JR

    (2004) Coccolithophores

    Springer.

    https://doi.org/10.1007/978-3-662-06278-4

    • Google Scholar
64. 1. Vardi A
    2. Van Mooy BAS
    3. Fredricks HF
    4. Popendorf KJ
    5. Ossolinski JE
    6. Haramaty L
    7. Bidle KD

    (2009) Viral glycosphingolipids induce lytic infection and cell death in marine phytoplankton

    Science 326:861–865.

    https://doi.org/10.1126/science.1177322

    • PubMed
    • Google Scholar
65. 1. Villar E
    2. Vannier T
    3. Vernette C
    4. Lescot M
    5. Cuenca M
    6. Alexandre A
    7. Bachelerie P
    8. Rosnet T
    9. Pelletier E
    10. Sunagawa S
    11. Hingamp P

    (2018) The ocean gene atlas: exploring the biogeography of plankton genes online

    Nucleic Acids Research 46:W289–W295.

    https://doi.org/10.1093/nar/gky376

    • PubMed
    • Google Scholar
66. 1. Vogt A
    2. Wietek J
    3. Hegemann P

    (2013) Gloeobacter rhodopsin, limitation of proton pumping at high electrochemical load

    Biophysical Journal 105:2055–2063.

    https://doi.org/10.1016/j.bpj.2013.08.031

    • PubMed
    • Google Scholar
67. 1. Weinert T
    2. Skopintsev P
    3. James D
    4. Dworkowski F
    5. Panepucci E
    6. Kekilli D
    7. Furrer A
    8. Brünle S
    9. Mous S
    10. Ozerov D
    11. Nogly P
    12. Wang M
    13. Standfuss J

    (2019) Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

    Science 365:61–65.

    https://doi.org/10.1126/science.aaw8634

    • PubMed
    • Google Scholar
68. 1. Wood JN
    2. Bevan SJ
    3. Coote PR
    4. Dunn PM
    5. Harmar A
    6. Hogan P
    7. Latchman DS
    8. Morrison C
    9. Rougon G
    10. Theveniau M

    (1990) Novel cell lines display properties of nociceptive sensory neurons

    Proceedings. Biological Sciences 241:187–194.

    https://doi.org/10.1098/rspb.1990.0084

    • PubMed
    • Google Scholar
69. 1. Yamauchi Y
    2. Konno M
    3. Ito S
    4. Tsunoda SP
    5. Inoue K
    6. Kandori H

    (2017) Molecular properties of a DTD channelrhodopsin from guillardia theta

    Biophysics and Physicobiology 14:57–66.

    https://doi.org/10.2142/biophysico.14.0_57

    • PubMed
    • Google Scholar
70. 1. Zabelskii D
    2. Alekseev A
    3. Kovalev K
    4. Rankovic V
    5. Balandin T
    6. Soloviov D
    7. Bratanov D
    8. Savelyeva E
    9. Podolyak E
    10. Volkov D
    11. Vaganova S
    12. Astashkin R
    13. Chizhov I
    14. Yutin N
    15. Rulev M
    16. Popov A
    17. Eria-Oliveira AS
    18. Rokitskaya T
    19. Mager T
    20. Antonenko Y
    21. Rosselli R
    22. Armeev G
    23. Shaitan K
    24. Vivaudou M
    25. Büldt G
    26. Rogachev A
    27. Rodriguez-Valera F
    28. Kirpichnikov M
    29. Moser T
    30. Offenhäusser A
    31. Willbold D
    32. Koonin E
    33. Bamberg E
    34. Gordeliy V

    (2020) Viral rhodopsins 1 are an unique family of light-gated cation channels

    Nature Communications 11:1–16.

    https://doi.org/10.1038/s41467-020-19457-7

    • Google Scholar

Author details

1. Shoko Hososhima

   Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7465-2907

2. Ritsu Mizutori

   Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Rei Abe-Yoshizumi

   Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7082-7949

4. Andrey Rozenberg

   Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Shunta Shigemura

   Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

6. Alina Pushkarev

   Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel

   Present address

   Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany

   Contribution

   Conceptualization, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Masae Konno

   Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan

   Present address

   The Institute for Solid State Physics, The University of Tokyo, Kashiwa, Japan

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

8. Kota Katayama

   1. Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan
   2. OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Japan

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

9. Keiichi Inoue

   Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan

   Present address

   The Institute for Solid State Physics, The University of Tokyo, Kashiwa, Japan

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6898-4347

10. Satoshi P Tsunoda

    1. Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan
    2. OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Japan

    Contribution

    Investigation, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3636-1521

11. Oded Béjà

    Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6629-0192

12. Hideki Kandori

    1. Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Japan
    2. OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Japan

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - original draft

    For correspondence

    kandori@nitech.ac.jp

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4922-1344

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