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.

Proton-transporting heliorhodopsins from marine giant viruses.

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter. V2HeR3 absorbs blue-green light, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a PAG of the Schiff base, and mutations at these positions converted the protein into an outward proton pump. Three environmental viral HeRs from the same group as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tool, similarly to type-1 microbial rhodopsins.

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.

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.

Light-Induced Conformational Alterations in Heliorhodopsin Triggered by the Retinal Excited State.

Heliorhodopsins are a recently discovered diverse retinal protein family with an inverted topology of the opsin where the retinal protonated Schiff base proton is facing the cell cytoplasmic side in contrast to type 1 rhodopsins. To explore whether light-induced retinal double-bond isomerization is a prerequisite for triggering protein conformational alterations, we utilized the retinal oxime formation reaction and thermal denaturation of a native heliorhodopsin of Thermoplasmatales archaeon SG8-52-1 (TaHeR) as well as a trans-locked retinal analogue (TaHeRL) in which the critical C13═C14 double-bond isomerization is prevented. We found that both reactions are light-accelerated not only in the native but also in the "locked" pigment despite lacking any isomerization. It is suggested that light-induced charge redistribution in the retinal excited state polarizes the protein and triggers protein conformational perturbations that thermally decay in microseconds. The extracted activation energy and the frequency factor for both the reactions reveal that the light enhancement of TaHeR differs distinctly from the earlier studied type 1 microbial rhodopsins.

Discovery of a microbial rhodopsin that is the most stable in extreme environments.

Microbial rhodopsin is a retinal protein that functions as an ion pump, channel, and sensory transducer, as well as a light sensor, as in biosensors and biochips. Tara76 rhodopsin is a typical proton-pumping rhodopsin that exhibits strong stability against extreme pH, detergent, temperature, salt stress, and dehydration stress and even under dual and triple conditions. Tara76 rhodopsin has a thermal stability approximately 20 times higher than that of thermal rhodopsin at 80°C and is even stable at 85°C. Tara76 rhodopsin is also stable at pH 0.02 to 13 and exhibits strong resistance in detergent, including Triton X-100 and SDS. We tested the current flow that electrical current flow across dried proteins on the paper at high temperatures using an electrode device, which was measured stably from 25°C up to 120°C. These properties suggest that this Tara76 rhodopsin is suitable for many applications in the fields of bioengineering and biotechnology.

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.

mRNA dynamics and alternative conformations adopted under low and high arginine concentrations control polyamine biosynthesis in Salmonella.

Putrescine belongs to the large group of polyamines, an essential class of metabolites that exists throughout all kingdoms of life. The Salmonella speF gene encodes an inducible ornithine decarboxylase that produces putrescine from ornithine. Putrescine can be also synthesized from arginine in a parallel metabolic pathway. Here, we show that speF expression is controlled at multiple levels through regulatory elements contained in a long leader sequence. At the heart of this regulation is a short open reading frame, orf34, which is required for speF production. Translation of orf34 interferes with Rho-dependent transcription termination and helps to unfold an inhibitory RNA structure sequestering speF ribosome-binding site. Two consecutive arginine codons in the conserved domain of orf34 provide a third level of speF regulation. Uninterrupted translation of orf34 under conditions of high arginine allows the formation of a speF mRNA structure that is degraded by RNase G, whereas ribosome pausing at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is resistant to RNase G. Thus, the rate of ribosome progression during translation of the upstream ORF influences the dynamics of speF mRNA folding and putrescine production. The identification of orf34 and its regulatory functions provides evidence for the evolutionary conservation of ornithine decarboxylase regulatory elements and putrescine production.

Heliorhodopsins are absent in diderm (Gram-negative) bacteria: Some thoughts and possible implications for activity.

Microbial heliorhodopsins are a new type of rhodopsins, currently believed to engage in light sensing, with an opposite membrane topology compared to type-1 and type-2 rhodopsins. We determined heliorhodopsins presence/absence is monoderms and diderms representatives from the Tara Oceans and freshwater metagenomes as well as metagenome assembled genome collections. Heliorhodopsins are absent in diderms, confirming our previous observations in cultured Proteobacteria. We do not rule out the possibility that heliorhodopsins serve as light sensors. However, this does not easily explain their absence from diderms. Based on these observations, we speculate on the putative role of heliorhodopsins in light-driven transport of amphiphilic molecules.

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.

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.

The Use of a Chimeric Rhodopsin Vector for the Detection of New Proteorhodopsins Based on Color.

Student microbial ecology laboratory courses are often conducted as condensed courses in which theory and wet lab work are combined in a very intensive short time period. In last decades, the study of marine microbial ecology is increasingly reliant on molecular-based methods, and as a result many of the research projects conducted in such courses require sequencing that is often not available on site and may take more time than a typical course allows. In this work, we describe a protocol combining molecular and functional methods for analyzing proteorhodopsins (PRs), with visible results in only 4-5 days that do not rely on sequencing. PRs were discovered in oceanic surface waters two decades ago, and have since been observed in different marine environments and diverse taxa, including the abundant alphaproteobacterial SAR11 group. PR subgroups are currently known to absorb green and blue light, and their distribution was previously explained by prevailing light conditions - green pigments at the surface and blue pigments in deeper waters, as blue light travels deeper in the water column. To detect PR in environmental samples, we created a chimeric plasmid suitable for direct expression of PRs using PCR amplification and functional analysis in Escherichia coli cells. Using this assay, we discovered several exceptional cases of PRs whose phenotypes differed from those predicted based on sequence only, including a previously undescribed yellow-light absorbing PRs. We applied this assay in two 10-days marine microbiology courses and found it to greatly enhance students' laboratory experience, enabling them to gain rapid visual feedback and colorful reward for their work. Furthermore we expect this assay to promote the use of functional assays for the discovery of new rhodopsin variants.

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.

Functional metagenomic screen reveals new and diverse microbial rhodopsins.

Ion-translocating retinylidene rhodopsins are widely distributed among marine and freshwater microbes. The translocation is light-driven, contributing to the production of biochemical energy in diverse microbes. Until today, most microbial rhodopsins had been detected using bioinformatics based on homology to other rhodopsins. In the past decade, there has been increased interest in microbial rhodopsins in the field of optogenetics since microbial rhodopsins were found to be most useful in vertebrate neuronal systems. Here we report on a functional metagenomic assay for detecting microbial rhodopsins. Using an array of narrow pH electrodes and light-emitting diode illumination, we were able to screen a metagenomic fosmid library to detect diverse marine proteorhodopsins and an actinorhodopsin based solely on proton-pumping activity. Our assay therefore provides a rather simple phenotypic means to enrich our understanding of microbial rhodopsins without any prior knowledge of the genomic content of the environmental entities screened.