Oded Béjà

Microbial rhodopsins are light-sensitive proteins vital to various phototrophic and sensory processes in microorganisms. Xanthorhodopsins, with their dual chromophore system involving retinal and carotenoids, have been predominantly studied in the extreme halophilic bacterium Salinibacter ruber and in the early-branching thylakoid-less cyanobacterium Gloeobacter violaceus, where they facilitate light-driven outward proton pumping. However, their distribution, binding specificity, and ecological significance in cyanobacteria remain poorly understood. Here we report the incidence of xanthorhodopsin genes in cyanobacterial genomes and characterize psXR, a xanthorhodopsin from an uncultured Antarctic cyanobacterium from the filamentous family of Pseudanabaenaceae that binds a hydroxylated carotenoid antenna. Through bioinformatic, spectroscopic, functional and structural analyses, we determine the properties of psXR and potential physiological roles of cyanobacterial xanthorhodopsins. Our findings suggest xanthorhodopsins’ role in modulating light-harvesting efficiency in cyanobacteria, particularly in extreme environments. The antenna binding and associated structural changes likely provide selective advantages for adapting to polar light conditions such as prolonged low light intensities and spectral shifts, contributing to cyanobacterial survival in harsh habitats.

Marine picocyanobacteria are abundant photosynthetic organisms of global importance. They coexist in the ocean with cyanophages—viruses that infect cyanobacteria. Cyanophages carry many auxiliary metabolic genes acquired from their hosts that are thought to redirect host metabolism for the phage’s benefit^1,2,3,4,5. One such gene is nblA, which is present in multiple cyanophage families^2,6,7,8. Under nutrient deprivation cyanobacterial NblA is responsible for inducing proteolytic degradation of the phycobilisome^9,10,11, the large cyanobacterial photosynthetic light-harvesting complex. This increases the pool of amino acids available for essential tasks¹¹, serving as a survival mechanism¹². Ectopic expression of different cyanophage nblA genes results in host pigment protein degradation^6,8,13. However, the benefit of the virus-encoded NblA for cyanophages and the broader impact on the host are unclear. Here, using a recently developed genetic manipulation system for marine cyanophages¹⁴, we reveal that viral NblA significantly accelerates the cyanophage infection cycle, directs degradation of the host phycobilisome and other proteins, and reduces host photosynthetic light-harvesting efficiency. Metagenomic analysis revealed that cyanophages carrying nblA are widespread in the oceans and comprise 35% and 65% of oceanic T7-like cyanophages in surface and deep photic zones, respectively. Our results show a large benefit of NblA to the cyanophage, while it exerts a negative effect on the host photosynthetic apparatus and host photosynthesis. These findings suggest that cyanophage NblA has an adverse global impact on light harvesting by oceanic picocyanobacteria.

Microbial rhodopsins represent a diverse superfamily of light-sensitive seven-transmembrane proteins with expanding phylogenetic diversity driven by advances in metagenomics. Among these, schizorhodopsins constitute a divergent family originally identified as inward proton pumps from Promethearchaeota (Asgard archaea). Here, we report that in addition to archaeal schizorhodopsins, many members of the family originate from bacteria and detail a comprehensive biophysical characterization of novel schizorhodopsins from Antarctic Minisyncoccota (Patescibacteria) and cyanobacteria, designated as paSzR and psSzR, respectively. Both proteins function as light-driven inward proton pumps, as confirmed through pH measurements in Escherichia coli cells. Laser-flash photolysis experiments identified multiple photointermediates (K, L, and M) characteristic of microbial rhodopsin photocycles, though with slower turnover rates compared to archaeal schizorhodopsins. Site-directed mutagenesis of conserved residues in the third and sixth transmembrane helices demonstrates differential structural requirements between paSzR and psSzR. Our phylogenetic reconstruction reveals that most bacterial schizorhodopsins cluster in a single lineage distinct from archaeal variants. These findings significantly expand our understanding of microbial rhodopsin diversity and provide crucial insights into alternative molecular mechanisms for light-driven proton translocation, with implications for microbial ecology in extreme environments.

Apusomonads are sediment-dwelling bacterivorous protists that are sister to all Opisthokonta. They have been found to show a negative phototactic response to blue light, mediated by an as-yet unidentified photoreceptive system. Here, by screening available apusomonad omics data we found genes of a distinct group of microbial rhodopsins, apusomonad rhodopsins (ApuRs). ApuRs, heterologously expressed in mammalian cells, absorbed near-UV or violet light, suggesting that ApuRs could be involved in apusomonads’ photoavoidance response toward short-wavelength light. Electrophysiological measurements indicate that ApuRs are anion-selective rhodopsin channels which evolved independently of the family of channelrhodopsins widespread in other unicellular eukaryotes. In ApuRs, channel opening is triggered by photoisomerization of the retinal from its all- trans form to 13- cis and 11- cis forms. We found that intracellular proton transfer is involved in channel opening and determines the channel’s open/close kinetics. These findings expand our understanding of the photobiology of heterotrophic flagellates and demonstrate that UV-absorbing ApuRs are in fact the most blue-shifted rhodopsin channels known to date.

Channelrhodopsins (ChRs) are light-gated ion channels. These proteins are widely used in optogenetics to optically manipulate neural activity. However, manipulation using short-wavelength light to activate ChRs causes cell toxicity and is hampered by low tissue penetration. To overcome these difficulties, although several red-shifted ChR variants have been identified, further red-shift is required for more efficient and noninvasive neural control. While molecular screening of ChRs requires high-cost experiments, recent machine-learning-based protein functionality prediction enables more efficient selection of target proteins for characterization. Here, we constructed an elastic-net machine-learning model trained on 1,163 experimental data to predict the maximum absorption wavelength ( λ _max ) of uncharacterized ChRs. The model suggested several red-shifted candidates, and we identified a viral rhodopsin channel, ChR024, with the second-longest λ _max as a cation-conducting ChR ( λ _max = ∼578 nm) after Chrimson ( λ _max = ∼580 nm). This result demonstrates the high impact of ML on reducing the screening costs of functional proteins.

Despite extensive research on carotenoids and microbial rhodopsins in aquatic environments, a fundamental understanding of the binding requirements of carotenoids that serve as auxiliary light-harvesting antennas for rhodopsins is still lacking. Our recent discovery of 3-hydroxylated xanthophyll-binding proteorhodopsins and xanthorhodopsins prompted us to investigate the role of keto and hydroxy functional groups in carotenoid binding to rhodopsins and their influence on energy transfer to the retinal chromophore. In this study, we examined the binding of 12 carotenoids to rhodopsin Kin4B8 (a protein of the xanthorhodopsin family, GenBank: OP056329) and assessed the energy transfer between the carotenoid and the retinal chromophore. We found that 3-hydroxylated xanthophylls were the most effective light-harvesting antennas among the carotenoids studied. While 4-ketocarotenoids also bound to the protein, their energy transfer efficiency was significantly reduced. In contrast, the presence of a 4-hydroxy group or the substitution of the β-ionone ring by an ε-ionone ring completely prevented binding. Furthermore, mutagenesis studies of Kin4B8 suggest that specific residues play a key role in the selective binding of carotenoids. These findings provide valuable insights into the structural determinants of rhodopsin-carotenoid interactions, which may aid in predicting the recruitment of various carotenoid antennas by retinal proteins.

Microbial rhodopsins are light-sensitive proteins vital to various phototrophic and sensory processes in microorganisms. Xanthorhodopsins, with their dual chromophore system involving retinal and carotenoids, have been predominantly studied in the extreme halophilic bacterium Salinibacter ruber and in the primitive thylakoid-less cyanobacterium Gloeobacter violaceus , where they facilitate light-driven outward proton pumping and enhanced light-harvesting. However, their distribution, binding specificity, and ecological significance in cyanobacteria remain poorly understood. Here we report widespread xanthorhodopsin genes in cyanobacterial genomes and characterize a protein from an Antarctic filamentous cyanobacterium that uniquely binds hydroxylated lutein. Through bioinformatics, spectroscopic, functional and structural assays analyses, we determine the properties and ecological role of this cyanobacterial xanthorhodopsin. Our findings highlight xanthorhodopsins' role in modulating light-harvesting efficiency in cyanobacteria, particularly in extreme environments. The lutein binding and associated structural changes likely provide selective advantages for adapting to polar light conditions, contributing to cyanobacterial survival in harsh habitats.

Aquatic bacterial rhodopsin proton pumps harvest light energy for photoheterotrophic growth and are known to contain hydroxylated carotenoids that expand the wavelengths of light utilized, but these have not been characterized in marine archaea. Here, by combining a marine chromophore extract with purified archaeal rhodopsins identified in marine metagenomes, we show light energy transfer from diverse hydroxylated carotenoids to heimdallarchaeial rhodopsins (HeimdallRs) from uncultured marine planktonic members of ‘Candidatus Kariarchaeaceae’ (‘Candidatus Asgardarchaeota’). These light-harvesting antennas absorb in the blue-light range and transfer energy to the green-light-absorbing retinal chromophore within HeimdallRs, enabling the use of light that is otherwise unavailable to the rhodopsin. Furthermore, we show elevated proton pumping by the antennas in HeimdallRs under white-light illumination, which better simulates the light conditions encountered by these archaea in their natural habitats. Our results indicate that light-harvesting antennas in microbial rhodopsins exist in families beyond xanthorhodopsins and proteorhodopsins and are present in both marine bacteria and archaea.

Apusomonads are a clade of understudied sediment-dwelling bacterivorous protists sister to Opisthokonta. Recently, apusomonads have been found to show a negative phototactic response to blue light. Here, by screening available apusomonad ‘omics data we found genes of a distinct group of microbial rhodopsins, the ApuRs. ApuRs heterologously expressed in mammalian cells absorbed near-UV/violet or blue light, suggesting that ApuRs could be involved in apusomonads’ photoavoidance response. Electrophysiological measurements indicate that ApuRs are anion-selective rhodopsin channels which evolved independently of the family of channelrhodopsins widespread in other unicellular eukaryotes. Among the known rhodopsin channels, ApuRs collectively demonstrate the most blue-shifted absorption spectra. In ApuRs, the channel opening is triggered by photoisomerization of the retinal from its all- trans form to 13- cis and 11- cis forms. We found that intracellular proton transfer is involved in channel opening and determined the channel’s open/close kinetics. These findings expand our understanding of the photobiology of heterotrophic flagellates and showcase the significance of these organisms as a source of new rhodopsin families with unanticipated functions.

Marine picocyanobacteria are abundant photosynthetic organisms of global importance. They coexist in the ocean with cyanophages, viruses that infect cyanobacteria. Cyanophages carry many auxiliary metabolic genes acquired from their hosts that are thought to redirect host metabolism for the phage’s benefit ^1–5 . One such gene is nblA which is present in multiple cyanophage families ^2,6–8 . Under nutrient deprivation the cyanobacterial NblA is responsible for inducing proteolytic degradation of the phycobilisome ^9–11 , the large cyanobacterial photosynthetic light harvesting complex. This increases the pool of amino acids available for essential tasks ¹¹ , serving as a survival mechanism ¹² . Ectopic expression of different cyanophage nblA genes results in host pigment protein degradation ^9,6 . However, the benefit of the cyanophage-encoded NblA for the cyanophage and the broader impact on the host are unknown. Here, using a recently developed genetic manipulation system for marine cyanophages ¹⁴ , we reveal that cyanophage NblA significantly accelerates the cyanophage infection cycle, directs degradation of the host phycobilisome and other proteins and reduces host photosynthetic light harvesting efficiency. Metagenomic analysis revealed that cyanophages carrying nblA are widespread in the oceans and compose 35% and 65% of oceanic T7-like cyanophages in the surface and deep photic zones, respectively. Our results show a large benefit of the nblA gene to the cyanophage while exerting a negative effect on the host photosynthetic apparatus and host photosynthesis. These findings suggest that cyanophage nblA has an adverse global impact on light harvesting by oceanic picocyanobacteria.