Changes in microbial community structure related to biodegradation of eelgrass (Zostera marina).

Seagrass meadows produce organic carbon and deposit it on the seabed through the decaying process. Microbial activity is closely related to the process of eelgrass death and collapse. We investigated the microbial community structure of eelgrass during the eelgrass decomposition process by using a microcosm containing raw seawater and excised eelgrass leaves collected from a Zostera marina bed in Futtsu, Chiba Prefecture, Japan. The fast-growing microbes (i.e., Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia) rapidly adhered to the eelgrass leaf surface and proliferated in the first two weeks but gradually decreased the relative abundance as the months moved on. On the other hand, the slow-growing microbes (i.e., Cytophagia, Anaerolineae, Thaumarchaeota, and Actinobacteria) became predominant over the eelgrass surface late in the culture experiment (120, 180 days). The fast-growing groups of Gammaproteobacteria and Flavobacteriia appear to be closely related to the initial decomposition of eelgrass, especially the rapid decomposition of leaf-derived biopolymers. Changes in nitrogen content due to the bacterial rapid consumption of readily degradable organic carbon induced changes in the community structure at the early stage of eelgrass decomposition. In addition, shifts in the C/N ratio were driven by microbial community changes during later decomposition phases.

Draft genome sequences of three rhodopsin possessing Croceitalea sp. strains, isolated from the sea surface microlayer in Japan.

Here, we present the draft genome sequences of three Croceitalea sp. strains containing microbial rhodopsins, isolated from the Japanese coastal sea surface microlayer, which is exposed to intense sunlight. This study will contribute to the understanding of the genus Croceitalea and the diversity of microbial rhodopsins.

Particle-attached Microbes in Eelgrass Vegetation Areas Differ in Community Structure Depending on the Distance from the Eelgrass Bed.

Zostera marina (eelgrass) is a submerged flowering plant often found in the coastal areas of Japan. Large amounts of suspended particles form in highly productive environments, such as eelgrass beds, and the behavior of these particles is expected to affect the surrounding microbial community. We investigated the microbial community structure of suspended particles in three eelgrass fields (Ikuno-Shima Is., Mutsu Bay, and Nanao Bay) and inferred the formation and dynamics of suspended particles from a microbial community structure ana-lysis. Seawater samples were collected directly above each eelgrass bed (eelgrass-covering) and from locations dozens of meters away from the eelgrass bed (bare-ground). In consideration of the two different lifestyles of marine microbes, microbial communities were obtained from particle-attached (PA) and free-living (FL) states. Differences in microbial diversity and community structures were observed between PA and FL in all eelgrass beds. The FL microbial community was similar between the two sampling points (eelgrass-covering and bare-ground), whereas a significant difference was noted in the microbial community structure of suspended particles between the two sampling points. This difference appeared to be due to the supply of organic matter from the eelgrass sea ground and leaf-attached detritus produced by microbial activity. In addition, the classes Flavobacteriia, Alphaproteobacteria, and Gammaproteobacteria were abundant in the PA and FL fractions. Furthermore, many sequences of the key groups (e.g., Planctomycetes and Verrucomicrobia) were exclusively detected in the PA fraction, in which they may circulate nutrients. The present results provide insights into the microbial communities of suspended particles and provide the first step towards understanding their biogeochemical impact on the eelgrass bed.

Widespread use of proton-pumping rhodopsin in Antarctic phytoplankton.

Photosynthetic carbon (C) fixation by phytoplankton in the Southern Ocean (SO) plays a critical role in regulating air-sea exchange of carbon dioxide and thus global climate. In the SO, photosynthesis (PS) is often constrained by low iron, low temperatures, and low but highly variable light intensities. Recently, proton-pumping rhodopsins (PPRs) were identified in marine phytoplankton, providing an alternate iron-free, light-driven source of cellular energy. These proteins pump protons across cellular membranes through light absorption by the chromophore retinal, and the resulting pH energy gradient can then be used for active membrane transport or for synthesis of adenosine triphosphate. Here, we show that PPR is pervasive in Antarctic phytoplankton, especially in iron-limited regions. In a model SO diatom, we found that it was localized to the vacuolar membrane, making the vacuole a putative alternative phototrophic organelle for light-driven production of cellular energy. Unlike photosynthetic C fixation, which decreases substantially at colder temperatures, the proton transport activity of PPR was unaffected by decreasing temperature. Cellular PPR levels in cultured SO diatoms increased with decreasing iron concentrations and energy production from PPR photochemistry could substantially augment that of PS, especially under high light intensities, where PS is often photoinhibited. PPR gene expression and high retinal concentrations in phytoplankton in SO waters support its widespread use in polar environments. PPRs are an important adaptation of SO phytoplankton to growth and survival in their cold, iron-limited, and variable light environment.

Light-driven Proton Pumps as a Potential Regulator for Carbon Fixation in Marine Diatoms.

Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.

Distribution and survival strategies of endemic and cosmopolitan diazotrophs in the Arctic Ocean.

Dinitrogen (N2) fixation is the major source of reactive nitrogen in the ocean and has been considered to occur specifically in low-latitude oligotrophic oceans. Recent studies have shown that N2 fixation also occurs in the polar regions and thus is a global process, although the physiological and ecological characteristics of polar diazotrophs are not yet known. Here, we successfully reconstructed diazotroph genomes, including that of cyanobacterium UCYN-A (Candidatus 'Atelocyanobacterium thalassa'), from metagenome data corresponding to 111 samples isolated from the Arctic Ocean. These diazotrophs were highly abundant in the Arctic Ocean (max., 1.28% of the total microbial community), suggesting that they have important roles in the Arctic ecosystem and biogeochemical cycles. Further, we show that diazotrophs within genera Arcobacter, Psychromonas, and Oceanobacter are prevalent in the <0.2 µm fraction in the Arctic Ocean, indicating that current methods cannot capture their N2 fixation. Diazotrophs in the Arctic Ocean were either Arctic-endemic or cosmopolitan species from their global distribution patterns. Arctic-endemic diazotrophs, including Arctic UCYN-A, were similar to low-latitude-endemic and cosmopolitan diazotrophs in genome-wide function, however, they had unique gene sets (e.g., diverse aromatics degradation genes), suggesting adaptations to Arctic-specific conditions. Cosmopolitan diazotrophs were generally non-cyanobacteria and commonly had the gene that encodes the cold-inducible RNA chaperone, which presumably makes their survival possible even in deep, cold waters of global ocean and polar surface waters. This study shows global distribution pattern of diazotrophs with their genomes and provides clues to answering the question of how diazotrophs can inhabit polar waters.

A blue-shifted anion channelrhodopsin from the Colpodellida alga Vitrella brassicaformis.

Microbial rhodopsins, a family of photoreceptive membrane proteins containing the chromophore retinal, show a variety of light-dependent molecular functions. Channelrhodopsins work as light-gated ion channels and are widely utilized for optogenetics, which is a method for controlling neural activities by light. Since two cation channelrhodopsins were identified from the chlorophyte alga Chlamydomonas reinhardtii, recent advances in genomic research have revealed a wide variety of channelrhodopsins including anion channelrhodopsins (ACRs), describing their highly diversified molecular properties (e.g., spectral sensitivity, kinetics and ion selectivity). Here, we report two channelrhodopsin-like rhodopsins from the Colpodellida alga Vitrella brassicaformis, which are phylogenetically distinct from the known channelrhodopsins. Spectroscopic and electrophysiological analyses indicated that these rhodopsins are green- and blue-sensitive pigments (λmax = ~ 550 and ~ 440 nm) that exhibit light-dependent ion channeling activities. Detailed electrophysiological analysis revealed that one of them works as a monovalent anion (Cl-, Br- and NO3-) channel and we named it V. brassicaformis anion channelrhodopsin-2, VbACR2. Importantly, the absorption maximum of VbACR2 (~ 440 nm) is blue-shifted among the known ACRs. Thus, we identified the new blue-shifted ACR, which leads to the expansion of the molecular diversity of ACRs.

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.

Identification of a Functionally Efficient and Thermally Stable Outward Sodium-Pumping Rhodopsin (BeNaR) from a Thermophilic Bacterium.

Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na+), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Thus, we named the protein Bellilinea Na+-pumping rhodopsin, BeNaR. Of note, its Na+-pumping activity is significantly greater than that of the known Na+-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524 nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. From the results, we propose a model for the outward Na+-pumping mechanism of BeNaR. The efficient Na+-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools.

Comparison of species-specific qPCR and metabarcoding methods to detect small pelagic fish distribution from open ocean environmental DNA.

Environmental DNA (eDNA) is increasingly used to noninvasively monitor aquatic animals in freshwater and coastal areas. However, the use of eDNA in the open ocean (hereafter referred to OceanDNA) is still limited because of the sparse distribution of eDNA in the open ocean. Small pelagic fish have a large biomass and are widely distributed in the open ocean. We tested the performance of two OceanDNA analysis methods-species-specific qPCR (quantitative polymerase chain reaction) and MiFish metabarcoding using universal primers-to determine the distribution of small pelagic fish in the open ocean. We focused on six small pelagic fish species (Sardinops melanostictus, Engraulis japonicus, Scomber japonicus, Scomber australasicus, Trachurus japonicus, and Cololabis saira) and selected the Kuroshio Extension area as a testbed, because distribution of the selected species is known to be influenced by the strong frontal structure. The results from OceanDNA methods were compared to those of net sampling to test for consistency. Then, we compared the detection performance in each target fish between the using of qPCR and MiFish methods. A positive correlation was evident between the qPCR and MiFish detection results. In the ranking of the species detection rates and spatial distribution estimations, comparable similarity was observed between results derived from the qPCR and MiFish methods. In contrast, the detection rate using the qPCR method was always higher than that of the MiFish method. Amplification bias on non-target DNA and low sample DNA quantity seemed to partially result in a lower detection rate for the MiFish method; the reason is still unclear. Considering the ability of MiFish to detect large numbers of species and the quantitative nature of qPCR, the combined usage of the two methods to monitor quantitative distribution of small pelagic fish species with information of fish community structures was recommended.

The OceanDNA MAG catalog contains over 50,000 prokaryotic genomes originated from various marine environments.

Marine microorganisms are immensely diverse and play fundamental roles in global geochemical cycling. Recent metagenome-assembled genome studies, with particular attention to large-scale projects such as Tara Oceans, have expanded the genomic repertoire of marine microorganisms. However, published marine metagenome data is still underexplored. We collected 2,057 marine metagenomes covering various marine environments and developed a new genome reconstruction pipeline. We reconstructed 52,325 qualified genomes composed of 8,466 prokaryotic species-level clusters spanning 59 phyla, including genomes from the deep-sea characterized as deeper than 1,000 m (n = 3,337), low-oxygen zones of <90 µmol O2 per kg water (n = 7,884), and polar regions (n = 7,752). Novelty evaluation using a genome taxonomy database shows that 6,256 species (73.9%) are novel and include genomes of high taxonomic novelty, such as new class candidates. These genomes collectively expanded the known phylogenetic diversity of marine prokaryotes by 34.2%, and the species representatives cover 26.5-42.0% of prokaryote-enriched metagenomes. Thoroughly leveraging accumulated metagenomic data, this genome resource, named the OceanDNA MAG catalog, illuminates uncharacterized marine microbial 'dark matter' lineages.

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers.

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Microbial communities on eelgrass (Zostera marina) thriving in Tokyo Bay and the possible source of leaf-attached microbes.

Zostera marina (eelgrass) is classified as one of the marine angiosperms and is widely distributed throughout much of the Northern Hemisphere. The present study investigated the microbial community structure and diversity of Z. marina growing in Futtsu bathing water, Chiba prefecture, Japan. The purpose of this study was to provide new insight into the colonization of eelgrass leaves by microbial communities based on leaf age and to compare these communities to the root-rhizome of Z. marina, and the surrounding microenvironments (suspended particles, seawater, and sediment). The microbial composition of each sample was analyzed using 16S ribosomal gene amplicon sequencing. Each sample type was found to have a unique microbial community structure. Leaf-attached microbes changed in their composition depending on the relative age of the eelgrass leaf. Special attention was given to a potential microbial source of leaf-attached microbes. Microbial communities of marine particles looked more like those of eelgrass leaves than those of water samples. This finding suggests that leaf-attached microbes were derived from suspended particles, which could allow them to go back and forth between eelgrass leaves and the water column.

Diversity and Composition of Microbial Communities in an Eelgrass (Zostera marina) Bed in Tokyo Bay, Japan.

Zostera marina (eelgrass) is a widespread seagrass species that forms diverse and productive habitats along coast lines throughout much of the northern hemisphere. The present study investigated the microbial consortia of Z. marina growing at Futtsu clam-digging beach, Chiba prefecture, Japan. The following environmental samples were collected: sediment, seawater, plant leaves, and the root-rhizome. Sediment and seawater samples were obtained from three sampling points: inside, outside, and at the marginal point of the eelgrass bed. The microbial composition of each sample was analyzed using 16S ribosomal gene amplicon sequencing. Microbial communities on the dead (withered) leaf surface markedly differed from those in sediment, but were similar to those in seawater. Eelgrass leaves and surrounding seawater were dominated by the bacterial taxa Rhodobacterales (Alphaproteobacteria), whereas Rhodobacterales were a minor group in eelgrass sediment. Additionally, we speculated that the order Sphingomonadales (Alphaproteobacteria) acts as a major degrader during the decomposition process and constantly degrades eelgrass leaves, which then spread into the surrounding seawater. Withered eelgrass leaves did not accumulate on the surface sediment because they were transported out of the eelgrass bed by wind and residual currents unique to the central part of Tokyo Bay.

Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization.

Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift.

The photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named 'schizorhodopsin (SzR)' has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pKa = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pKa = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral 'blue-shift' due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

Bacterium Lacking a Known Gene for Retinal Biosynthesis Constructs Functional Rhodopsins.

Microbial rhodopsins, comprising a protein moiety (rhodopsin apoprotein) bound to the light-absorbing chromophore retinal, function as ion pumps, ion channels, or light sensors. However, recent genomic and metagenomic surveys showed that some rhodopsin-possessing prokaryotes lack the known genes for retinal biosynthesis. Since rhodopsin apoproteins cannot absorb light energy, rhodopsins produced by prokaryotic strains lacking genes for retinal biosynthesis are hypothesized to be non-functional in cells. In the present study, we investigated whether Aurantimicrobium minutum KNCT, which is widely distributed in terrestrial environments and lacks any previously identified retinal biosynthesis genes, possesses functional rhodopsin. We initially measured ion transport activity in cultured cells. A light-induced pH change in a cell suspension of rhodopsin-possessing bacteria was detected in the absence of exogenous retinal. Furthermore, spectroscopic analyses of the cell lysate and HPLC-MS/MS analyses revealed that this strain contained an endogenous retinal. These results confirmed that A. minutum KNCT possesses functional rhodopsin and, hence, produces retinal via an unknown biosynthetic pathway. These results suggest that rhodopsin-possessing prokaryotes lacking known retinal biosynthesis genes also have functional rhodopsins.

A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage.

Microbial rhodopsin is a photoreceptor protein found in various bacteria and archaea, and it is considered to be a light-utilization device unique to heterotrophs. Recent studies have shown that several cyanobacterial genomes also include genes that encode rhodopsins, indicating that these auxiliary light-utilizing proteins may have evolved within photoautotroph lineages. To explore this possibility, we performed a large-scale genomic survey to clarify the distribution of rhodopsin and its phylogeny. Our surveys revealed a novel rhodopsin clade, cyanorhodopsin (CyR), that is unique to cyanobacteria. Genomic analysis revealed that rhodopsin genes show a habitat-biased distribution in cyanobacterial taxa, and that the CyR clade is composed exclusively of non-marine cyanobacterial strains. Functional analysis using a heterologous expression system revealed that CyRs function as light-driven outward H+ pumps. Examination of the photochemical properties and crystal structure (2.65 Å resolution) of a representative CyR protein, N2098R from Calothrix sp. NIES-2098, revealed that the structure of the protein is very similar to that of other rhodopsins such as bacteriorhodopsin, but that its retinal configuration and spectroscopic characteristics (absorption maximum and photocycle) are distinct from those of bacteriorhodopsin. These results suggest that the CyR clade proteins evolved together with chlorophyll-based photosynthesis systems and may have been optimized for the cyanobacterial environment.

Vectorial Proton Transport Mechanism of RxR, a Phylogenetically Distinct and Thermally Stable Microbial Rhodopsin.

Rubrobacter xylanophilus rhodopsin (RxR) is a phylogenetically distinct and thermally stable seven-transmembrane protein that functions as a light-driven proton (H+) pump with the chromophore retinal. To characterize its vectorial proton transport mechanism, mutational and theoretical investigations were performed for carboxylates in the transmembrane region of RxR and the sequential proton transport steps were revealed as follows: (i) a proton of the retinylidene Schiff base (Lys209) is transferred to the counterion Asp74 upon formation of the blue-shifted M-intermediate in collaboration with Asp205, and simultaneously, a respective proton is released from the proton releasing group (Glu187/Glu197) to the extracellular side, (ii) a proton of Asp85 is transferred to the Schiff base during M-decay, (iii) a proton is taken up from the intracellular side to Asp85 during decay of the red-shifted O-intermediate. This ion transport mechanism of RxR provides valuable information to understand other ion transporters since carboxylates are generally essential for their functions.