Phototrophy by antenna-containing rhodopsin pumps in aquatic environments.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

Microbial Rhodopsins: The Last Two Decades.

Microbial rhodopsins are diverse photoreceptive proteins containing a retinal chromophore and are found in all domains of cellular life and are even encoded in genomes of viruses. These rhodopsins make up two families: type 1 rhodopsins and the recently discovered heliorhodopsins. These families have seven transmembrane helices with similar structures but opposing membrane orientation. Microbial rhodopsins participate in a portfolio of light-driven energy and sensory transduction processes. In this review we present data collected over the last two decades about these rhodopsins and describe their diversity, functions, and biological and ecological roles.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

A distinct abundant group of microbial rhodopsins discovered using functional metagenomics.

Many organisms capture or sense sunlight using rhodopsin pigments1,2, which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families 1 , type-1 (microbial rhodopsins) and type-2 (animal rhodopsins). The two families share similar topologies and contain seven transmembrane helices that form a pocket in which retinal is linked covalently as a protonated Schiff base to a lysine at the seventh transmembrane helix2,3. Type-1 and type-2 rhodopsins show little or no sequence similarity to each other, as a consequence of extensive divergence from a common ancestor or convergent evolution of similar structures 1 . Here we report a previously unknown and diverse family of rhodopsins-which we term the heliorhodopsins-that we identified using functional metagenomics and that are distantly related to type-1 rhodopsins. Heliorhodopsins are embedded in the membrane with their N termini facing the cell cytoplasm, an orientation that is opposite to that of type-1 or type-2 rhodopsins. Heliorhodopsins show photocycles that are longer than one second, which is suggestive of light-sensory activity. Heliorhodopsin photocycles accompany retinal isomerization and proton transfer, as in type-1 and type-2 rhodopsins, but protons are never released from the protein, even transiently. Heliorhodopsins are abundant and distributed globally; we detected them in Archaea, Bacteria, Eukarya and their viruses. Our findings reveal a previously unknown family of light-sensing rhodopsins that are widespread in the microbial world.

Seasonal and diel patterns of abundance and activity of viruses in the Red Sea.

Virus-microbe interactions have been studied in great molecular details for many years in cultured model systems, yielding a plethora of knowledge on how viruses use and manipulate host machinery. Since the advent of molecular techniques and high-throughput sequencing, methods such as cooccurrence, nucleotide composition, and other statistical frameworks have been widely used to infer virus-microbe interactions, overcoming the limitations of culturing methods. However, their accuracy and relevance is still debatable as cooccurrence does not necessarily mean interaction. Here we introduce an ecological perspective of marine viral communities and potential interaction with their hosts, using analyses that make no prior assumptions on specific virus-host pairs. By size fractionating water samples into free viruses and microbes (i.e., also viruses inside or attached to their hosts) and looking at how viral group abundance changes over time along both fractions, we show that the viral community is undergoing a change in rank abundance across seasons, suggesting a seasonal succession of viruses in the Red Sea. We use abundance patterns in the different size fractions to classify viral clusters, indicating potential diverse interactions with their hosts and potential differences in life history traits between major viral groups. Finally, we show hourly resolved variations of intracellular abundance of similar viral groups, which might indicate differences in their infection cycles or metabolic capacities.

Diverse heliorhodopsins detected via functional metagenomics in freshwater Actinobacteria, Chloroflexi and Archaea.

The recently discovered rhodopsin family of heliorhodopsins (HeRs) is abundant in diverse microbial environments. So far, the functional and biological roles of HeRs remain unknown. To tackle this issue, we combined experimental and computational screens to gain some novel insights. Here, 10 readily expressed HeR genes were found using functional metagenomics on samples from two freshwater environments. These HeRs originated from diverse prokaryotic groups: Actinobacteria, Chloroflexi and Archaea. Heterologously expressed HeRs absorbed light in the green and yellow wavelengths (543-562 nm) and their photocycles exhibited diverse kinetic characteristics. To approach the physiological function of the HeRs, we used our environmental clones along with thousands of microbial genomes to analyze genes neighbouring HeRs. The strongest association was found with the DegV family involved in activation of fatty acids, which allowed us to hypothesize that HeRs might be involved in light-induced membrane lipid modifications.

TAT Rhodopsin Is an Ultraviolet-Dependent Environmental pH Sensor.

In many rhodopsins, the retinal Schiff base pKa remains very high, ensuring Schiff base protonation captures visible light. Nevertheless, recently we found that TAT rhodopsin contains protonated and unprotonated forms at physiological pH. The protonated form displays a unique photochemical behavior in which the primary K intermediate returns to the original state within 10-5 s, and the lack of photocycle completion poses questions about the functional role of TAT rhodopsin. Here we studied the molecular properties of the protonated and unprotonated forms of the Schiff base in TAT rhodopsin. We confirmed no photointermediate formation at >10-5 s for the protonated form of TAT rhodopsin in microenvironments such as detergents, nanodiscs, and liposomes. In contrast, the unprotonated form features a very long photocycle with a time constant of 15 s. A low-temperature study revealed that the primary reaction of the unprotonated form is all-trans to 13-cis photoisomerization, which is usual, but with a proton transfer reaction occurring at 77 K, which is unusual. The active intermediate contains the unprotonated Schiff base as well as the resting state. Electrophysiological measurements excluded ion-transport activity for TAT rhodopsin, while transient outward proton movement only at an alkaline extracellular pH indicates that TAT rhodopsin senses the extracellular pH. On the basis of the findings presented here, we propose that TAT rhodopsin is an ultraviolet (UV)-dependent environmental pH sensor in marine bacteria. At acidic pH, absorbed visible light energy is quickly dissipated into heat without any function. In contrast, when the environmental pH becomes high, absorption of UV/blue light yields formation of the long-lived intermediates, possibly driving the signal transduction cascade in marine bacteria.

Anion binding to mutants of the Schiff base counterion in heliorhodopsin 48C12.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, contains a single counterion, E107, which is specific to HeR 48C12. In this paper, we examined possible anion binding into the wild-type (WT) and E107 mutants of HeR 48C12. We prepared E107A, E107Q, and E107D, and chloride binding was tested by measuring absorption spectra using UV-visible spectroscopy under various anionic conditions. The experimental results clearly showed no anion binding to WT and E107D, where E107 and D107 acted as the Schiff base counterion, respectively. On the other hand, anion binding was observed to the Schiff base region in E107A and E107Q. In the case of E107A, λmax was 553, 559, 565, and 549 nm for Cl-, Br-, I-, and NO3-, respectively. Similar halide-size dependence on the absorption spectra of the chromophore in solution strongly suggests that the anion acted as the direct hydrogen-bonding acceptor of the protonated Schiff base in E107A. In the case of E107Q, λmax was 577, 578, 579, and 581 nm for Cl-, Br-, I-, and NO3-, respectively. Based on the small halide dependence, we interpreted the C[double bond, length as m-dash]O group of the Q107 side chain as the hydrogen-bonding acceptor. Moreover, the anion stabilized the protonated state without a direct hydrogen bond with the Schiff base. Structures around the Schiff base region are discussed for the WT and E107 mutants of HeR 48C12.

Mutation Study of Heliorhodopsin 48C12.

Rhodopsins are heptahelical transmembrane photoactive protein families: type 1 (microbial rhodopsins) and type 2 (animal rhodopsins). Both families share similar topologies and chromophore retinal, which is linked covalently as a protonated Schiff base to a Lys at the transmembrane 7 helix. Recently, through functional metagenomics analysis, we reported an unnoticed diverse family, heliorhodopsins (HeRs), which are abundant and distributed globally in archaea, bacteria, eukarya, and viruses. The sequence identity is <15% between HeRs and type 1 rhodopsins, so that many aspects of the molecular properties of HeRs remain unknown. Herein, to gain information about the residues responsible for the interaction with the chromophore, we applied Ala scanning to 30 candidate residues in HeR 48C12. As a result, 12 mutants showed no absorption change, eight exhibited a spectral blue-shift, six exhibited a spectral red-shift, and four did not form a pigment. R104, Y108, G145, and K241 play crucial roles in pigment formation. A combination of single mutants successfully engineered pigments absorbing at 523 nm (S112A/M141A) and 571 nm (H80A/S237A), covering more than ∼50 nm. These results provide fundamental knowledge about the molecular properties of HeRs.

Functional marine metagenomic screening for anti-quorum sensing and anti-biofilm activity.

Quorum sensing (QS), a cell-to-cell communication process, entails the production of signaling molecules that enable synchronized gene expression in microbial communities to regulate myriad microbial functions, including biofilm formation. QS disruption may constitute an innovative approach to the design of novel antifouling and anti-biofilm agents. To identify novel quorum sensing inhibitors (QSI), 2,500 environmental bacterial artificial chromosomes (BAC) from uncultured marine planktonic bacteria were screened for QSI activity using soft agar overlaid with wild type Chromobacterium violaceum as an indicator. Of the BAC library clones, 7% showed high QSI activity (>40%) against the indicator bacterium, suggesting that QSI is common in the marine environment. The most active compound, eluted from BAC clone 14-A5, disrupted QS signaling pathways and reduced biofilm formation in both Pseudomonas aeruginosa and Acinetobacter baumannii. The mass spectra of the active BAC clone (14-A5) that had been visualized by thin layer chromatography was dominated by a m/z peak of 362.1.

New biosynthetic pathway for pink pigments from uncultured oceanic viruses.

The pink open-chain tetrapyrrole pigment phycoerythrobilin (PEB) is employed by marine cyanobacteria, red algae and cryptophytes as a light-harvesting chromophore in phycobiliproteins. Genes encoding biosynthesis proteins for PEB have also been discovered in cyanophages, viruses that infect cyanobacteria, and mimic host pigment biosynthesis with the exception of PebS which combines the enzymatic activities of two host enzymes. In this study, we have identified novel members of the PEB biosynthetic enzyme families, heme oxygenases and ferredoxin-dependent bilin reductases. Encoding genes were found in metagenomic datasets and could be traced back to bacteriophage but not cyanophage origin. While the heme oxygenase exhibited standard activity, a new bilin reductase with highest homology to the teal pigment producing enzyme PcyA revealed PEB biosynthetic activity. Although PcyX possesses PebS-like activity both enzymes share only 9% sequence identity and likely catalyze the reaction via two independent mechanisms. Our data point towards the presence of phycobilin biosynthetic genes in phages that probably infect alphaproteobacteria and, therefore, further support a role of phycobilins outside oxygenic phototrophs.

Closing the gaps on the viral photosystem-I†psaDCAB gene organization.

Marine photosynthesis is largely driven by cyanobacteria, namely Synechococcus and Prochlorococcus. Genes encoding for photosystem (PS) I and II reaction centre proteins are found in cyanophages and are believed to increase their fitness. Two viral PSI gene arrangements are known, psaJF→C→A→B→K→E→D and psaD→C→A→B. The shared genes between these gene cassettes and their encoded proteins are distinguished by %G + C and protein sequence respectively. The data on the psaD→C→A→B gene organization were reported from only two partial gene cassettes coming from Global Ocean Sampling stations in the Pacific and Indian oceans. Now we have extended our search to 370 marine stations from six metagenomic projects. Genes corresponding to both PSI gene arrangements were detected in the Pacific, Indian and Atlantic oceans, confined to a strip along the equator (30°N and 30°S). In addition, we found that the predicted structure of the viral PsaA protein from the psaD→C→A→B organization contains a lumenal loop conserved in PsaA proteins from Synechococcus, but is completely absent in viral PsaA proteins from the psaJF→C→A→B→K→E→D gene organization and most Prochlorococcus strains. This may indicate a co-evolutionary scenario where cyanophages containing either of these gene organizations infect cyanobacterial ecotypes biogeographically restricted to the 30°N and 30°S equatorial strip.

Photosystem I gene cassettes are present in marine virus genomes.

Cyanobacteria of the Synechococcus and Prochlorococcus genera are important contributors to photosynthetic productivity in the open oceans. Recently, core photosystem II (PSII) genes were identified in cyanophages and proposed to function in photosynthesis and in increasing viral fitness by supplementing the host production of these proteins. Here we show evidence for the presence of photosystem I (PSI) genes in the genomes of viruses that infect these marine cyanobacteria, using pre-existing metagenomic data from the global ocean sampling expedition as well as from viral biomes. The seven cyanobacterial core PSI genes identified in this study, psaA, B, C, D, E, K and a unique J and F fusion, form a cluster in cyanophage genomes, suggestive of selection for a distinct function in the virus life cycle. The existence of this PSI cluster was confirmed with overlapping and long polymerase chain reaction on environmental DNA from the Northern Line Islands. Potentially, the seven proteins encoded by the viral genes are sufficient to form an intact monomeric PSI complex. Projection of viral predicted peptides on the cyanobacterial PSI crystal structure suggested that the viral-PSI components might provide a unique way of funnelling reducing power from respiratory and other electron transfer chains to the PSI.

Multiple Roles of a Conserved Glutamate Residue for Unique Biophysical Properties in a New Group of Microbial Rhodopsins Homologous to TAT Rhodopsin.

TAT rhodopsin, a microbial rhodopsin found in the marine SAR11 bacterium HIMB114, uniquely possesses a Thr-Ala-Thr (TAT) motif in the third transmembrane helix. Because of a low pKa value of the retinal Schiff base (RSB), TAT rhodopsin exhibits both a visible light-absorbing state with the protonated RSB and a UV-absorbing state with the deprotonated RSB at a neutral pH. The UV-absorbing state, in contrast to the visible light-absorbing one, converts to a long-lived photointermediate upon light absorption, implying that TAT rhodopsin functions as a pH-dependent light sensor. Despite detailed biophysical characterization and mechanistic studies on the TAT rhodopsin, it has been unknown whether other proteins with similarly unusual features exist. Here, we identified several new rhodopsin genes homologous to the TAT rhodopsin of HIMB114 (TATHIMB) from metagenomic data. Based on the absorption spectra of expressed proteins from these genes with visible and UV peaks similar to that of TATHIMB, they were classified as Twin-peaked Rhodopsin (TwR) family. TwR genes form a gene cluster with a set of 13 ORFs conserved in subclade IIIa of SAR11 bacteria. A glutamic acid in the second transmembrane helix, Glu54, is conserved in all of the TwRs. We investigated E54Q mutants of two TwRs and revealed that Glu54 plays critical roles in regulating the RSB pKa, oligomer formation, and the efficient photoreaction of the UV-absorbing state. The discovery of novel TwRs enables us to study the universality and individuality of the characteristics revealed so far in the original TATHIMB and contributes to further studies on mechanisms of unique properties of TwRs.

Isolation and infection cycle of a polinton-like virus virophage in an abundant marine alga.

Virophages are small double stranded DNA (dsDNA) viruses that can only replicate in a host by co-infecting with another virus. Marine algae are commonly associated with virophage-like elements such as Polinton-like viruses (PLVs) that remain largely uncharacterized. Here we isolated a PLV that co-infects the alga Phaeocystis globosa with the Phaeocystis globosa virus-14T (PgV-14T), a close relative of the "Phaeocystis globosa virus-virophage" genomic sequence. We name this PLV 'Gezel-14T. Gezel is phylogenetically distinct from the Lavidaviridae family where all known virophages belong. Gezel-14T co-infection decreases the fitness of its viral host by reducing burst sizes of PgV-14T, yet insufficiently to spare the cellular host population. Genomic screens show Gezel-14T-like PLVs integrated into Phaeocystis genomes, suggesting that these widespread viruses are capable of integration into cellular host genomes. This system presents an opportunity to better understand the evolution of eukaryotic dsDNA viruses as well as the complex dynamics and implications of viral parasitism.

Rhodopsin-mediated nutrient uptake by cultivated photoheterotrophic Verrucomicrobiota.

Rhodopsin photosystems convert light energy into electrochemical gradients used by the cell to produce ATP, or for other energy-demanding processes. While these photosystems are widespread in the ocean and have been identified in diverse microbial taxonomic groups, their physiological role in vivo has only been studied in few marine bacterial strains. Recent metagenomic studies revealed the presence of rhodopsin genes in the understudied Verrucomicrobiota phylum, yet their distribution within different Verrucomicrobiota lineages, their diversity, and function remain unknown. In this study, we show that more than 7% of Verrucomicrobiota genomes (n = 2916) harbor rhodopsins of different types. Furthermore, we describe the first two cultivated rhodopsin-containing strains, one harboring a proteorhodopsin gene and the other a xanthorhodopsin gene, allowing us to characterize their physiology under laboratory-controlled conditions. The strains were isolated in a previous study from the Eastern Mediterranean Sea and read mapping of 16S rRNA gene amplicons showed the highest abundances of these strains at the deep chlorophyll maximum (source of their inoculum) in winter and spring, with a substantial decrease in summer. Genomic analysis of the isolates suggests that motility and degradation of organic material, both energy demanding functions, may be supported by rhodopsin phototrophy in Verrucomicrobiota. Under culture conditions, we show that rhodopsin phototrophy occurs under carbon starvation, with light-mediated energy generation supporting sugar transport into the cells. Overall, this study suggests that photoheterotrophic Verrucomicrobiota may occupy an ecological niche where energy harvested from light enables bacterial motility toward organic matter and supports nutrient uptake.

Microbial rhodopsins on leaf surfaces of terrestrial plants.

The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface between the terrestrial biosphere and solar radiation. It is estimated to host up to 10(26) microbial cells that may intercept part of the photon flux impinging on the leaves. Based on 454-pyrosequencing-generated metagenome data, we report on the existence of diverse microbial rhodopsins in five distinct phyllospheres from tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Our findings, for the first time describing microbial rhodopsins from non-aquatic habitats, point towards the potential coexistence of microbial rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.

Searching Metagenomes for New Rhodopsins.

Most microbial groups have not been cultivated yet, and the only way to approach the enormous diversity of rhodopsins that they contain in a sensible timeframe is through the analysis of their genomes. High-throughput sequencing technologies have allowed the release of community genomics (metagenomics) of many habitats in the photic zones of the ocean and lakes. Already the harvest is impressive and included from the first bacterial rhodopsin (proteorhodopsin) to the recent discovery of heliorhodopsin by functional metagenomics. However, the search continues using bioinformatic or biochemical routes.

Saccharibacteria harness light energy using type-1 rhodopsins that may rely on retinal sourced from microbial hosts.

Microbial rhodopsins are a family of photoreceptive membrane proteins with a wide distribution across the Tree of Life. Within the candidate phyla radiation (CPR), a diverse group of putatively episymbiotic bacteria, the genetic potential to produce rhodopsins appears to be confined to a small clade of organisms from sunlit environments. Here, we characterize the metabolic context and biophysical features of Saccharibacteria Type-1 rhodopsin sequences derived from metagenomic surveys and show that these proteins function as outward proton pumps. This provides one of the only known mechanisms by which CPR can generate a proton gradient for ATP synthesis. These Saccharibacteria do not encode the genetic machinery to produce all-trans-retinal, the chromophore essential for rhodopsin function, but their rhodopsins are able to rapidly uptake this cofactor when provided in experimental assays. We found consistent evidence for the capacity to produce retinal from ß-carotene in microorganisms co-occurring with Saccharibacteria, and this genetic potential was dominated by members of the Actinobacteria, which are known hosts of Saccharibacteria in other habitats. If Actinobacteria serve as hosts for Saccharibacteria in freshwater environments, exchange of retinal for use by rhodopsin may be a feature of their associations.