Shared features and reciprocal complementation of the Chlamydomonas and Arabidopsis microbiota.

Microscopic algae release organic compounds to the region immediately surrounding their cells, known as the phycosphere, constituting a niche for colonization by heterotrophic bacteria. These bacteria take up algal photoassimilates and provide beneficial functions to their host, in a process that resembles the establishment of microbial communities associated with the roots and rhizospheres of land plants. Here, we characterize the microbiota of the model alga Chlamydomonas reinhardtii and reveal extensive taxonomic and functional overlap with the root microbiota of land plants. Using synthetic communities derived from C. reinhardtii and Arabidopsis thaliana, we show that phycosphere and root bacteria assemble into taxonomically similar communities on either host. We show that provision of diffusible metabolites is not sufficient for phycosphere community establishment, which additionally requires physical proximity to the host. Our data suggest the existence of shared ecological principles driving the assembly of the A. thaliana root and C. reinhardtii phycosphere microbiota, despite the vast evolutionary distance between these two photosynthetic organisms.

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.

An uncultured marine cyanophage encodes an active phycobilisome proteolysis adaptor protein NblA.

Phycobilisomes (PBS) are large water-soluble membrane-associated complexes in cyanobacteria and some chloroplasts that serve as light-harvesting antennae for the photosynthetic apparatus. When deplete of nitrogen or sulphur, cyanobacteria readily degrade their phycobilisomes allowing the cell to replenish these vanishing nutrients. The key regulator in the degradation process is NblA, a small protein (∼6 kDa), which recruits proteases to the PBS. It was discovered previously that not only do cyanobacteria possess nblA genes but also that they are encoded by genomes of some freshwater cyanophages. A recent study, using assemblies from oceanic metagenomes, revealed genomes of a novel uncultured marine cyanophage lineage, representatives of which contain genes coding for the PBS degradation protein. Here, we examined the functionality of nblA-like genes from these marine cyanophages by testing them in a freshwater model cyanobacterial nblA knockout. One of the viral NblA variants could complement the non-bleaching phenotype and restore PBS degradation. Our findings reveal a functional NblA from a novel marine cyanophage lineage. Furthermore, we shed new light on the distribution of nblA genes in cyanobacteria and cyanophages.

MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins.

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. Here we describe a metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of optogenetic tools from metagenomic databases and enhance general understanding of ChR function.

A novel uncultured marine cyanophage lineage with lysogenic potential linked to a putative marine Synechococcus 'relic' prophage.

Marine cyanobacteria are important contributors to primary production in the ocean and their viruses (cyanophages) affect the ocean microbial communities. Despite reports of lysogeny in marine cyanobacteria, a genome sequence of such temperate cyanophages remains unknown although genomic analysis indicate potential for lysogeny in certain marine cyanophages. Using assemblies from Red Sea and Tara Oceans metagenomes, we recovered genomes of a novel uncultured marine cyanophage lineage, which contain, in addition to common cyanophage genes, a phycobilisome degradation protein NblA, an integrase and a split DNA polymerase. The DNA polymerase forms a monophyletic clade with a DNA polymerase from a genomic island in Synechococcus WH8016. The island contains a relic prophage that does not resemble any previously reported cyanophage but shares several genes with the newly identified cyanophages reported here. Metagenomic recruitment indicates that the novel cyanophages are widespread, albeit at low abundance. Here, we describe a novel potentially lysogenic cyanophage family, their abundance and distribution in the marine environment.

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.

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.

Cyanophage-encoded lipid desaturases: oceanic distribution, diversity and function.

Cyanobacteria are among the most abundant photosynthetic organisms in the oceans; viruses infecting cyanobacteria (cyanophages) can alter cyanobacterial populations, and therefore affect the local food web and global biochemical cycles. These phages carry auxiliary metabolic genes (AMGs), which rewire various metabolic pathways in the infected host cell, resulting in increased phage fitness. Coping with stress resulting from photodamage appears to be a central necessity of cyanophages, yet the overall mechanism is poorly understood. Here we report a novel, widespread cyanophage AMG, encoding a fatty acid desaturase (FAD), found in two genotypes with distinct geographical distribution. FADs are capable of modulating the fluidity of the host's membrane, a fundamental stress response in living cells. We show that both viral FAD (vFAD) families are Δ9 lipid desaturases, catalyzing the desaturation at carbon 9 in C16 fatty acid chains. In addition, we present a comprehensive fatty acid profiling for marine cyanobacteria, which suggests a unique desaturation pathway of medium- to long-chain fatty acids no longer than C16, in accordance with the vFAD activity. Our findings suggest that cyanophages are capable of fiddling with the infected host's membranes, possibly leading to increased photoprotection and potentially enhancing viral-encoded photosynthetic proteins, resulting in a new viral metabolic network.

A myovirus encoding both photosystem I and II proteins enhances cyclic electron flow in infected Prochlorococcus cells.

Cyanobacteria are important contributors to primary production in the open oceans. Over the past decade, various photosynthesis-related genes have been found in viruses that infect cyanobacteria (cyanophages). Although photosystem II (PSII) genes are common in both cultured cyanophages and environmental samples 1-4 , viral photosystem I (vPSI) genes have so far only been detected in environmental samples 5,6 . Here, we have used a targeted strategy to isolate a cyanophage from the tropical Pacific Ocean that carries a PSI gene cassette with seven distinct PSI genes (psaJF, C, A, B, K, E, D) as well as two PSII genes (psbA, D). This cyanophage, P-TIM68, belongs to the T4-like myoviruses, has a prolate capsid, a long contractile tail and infects Prochlorococcus sp. strain MIT9515. Phage photosynthesis genes from both photosystems are expressed during infection, and the resultant proteins are incorporated into membranes of the infected host. Moreover, photosynthetic capacity in the cell is maintained throughout the infection cycle with enhancement of cyclic electron flow around PSI. Analysis of metagenomic data from the Tara Oceans expedition 7 shows that phages carrying PSI gene cassettes are abundant in the tropical Pacific Ocean, composing up to 28% of T4-like cyanomyophages. They are also present in the tropical Indian and Atlantic Oceans. P-TIM68 populations, specifically, compose on average 22% of the PSI-gene-cassette carrying phages. Our results suggest that cyanophages carrying PSI and PSII genes are likely to maintain and even manipulate photosynthesis during infection of their Prochlorococcus hosts in the tropical oceans.

Novel Abundant Oceanic Viruses of Uncultured Marine Group II Euryarchaeota.

Marine group II Euryarchaeota (MG-II) are among the most abundant microbes in oceanic surface waters [1-4]. So far, however, representatives of MG-II have not been cultivated, and no viruses infecting these organisms have been described. Here, we present complete genomes for three distinct groups of viruses assembled from metagenomic sequence datasets highly enriched for MG-II. These novel viruses, which we denote magroviruses, possess double-stranded DNA genomes of 65 to 100 kilobases in size that encode a structural module characteristic of head-tailed viruses and, unusually for archaeal and bacterial viruses, a nearly complete replication apparatus of apparent archaeal origin. The newly identified magroviruses are widespread and abundant and therefore are likely to be major ecological agents.

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.

Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii.

Chloroplast transformation in the photosynthetic alga Chlamydomonas reinhardtii has been used to explore the potential to use it as an inexpensive and easily scalable system for the production of therapeutic recombinant proteins. Diverse proteins, such as bacterial and viral antigens, antibodies and, immunotoxins have been successfully expressed in the chloroplast using endogenous and chimeric promoter sequences. In some cases, proteins have accumulated to high level, demonstrating that this technology could compete with current production platforms. This review focuses on the works that have engineered the chloroplast of C. reinhardtii with the aim of producing recombinant proteins intended for therapeutical use in humans or animals.