Phenology and ecological role of aerobic anoxygenic phototrophs in freshwaters.

BACKGROUND: Aerobic anoxygenic phototrophic (AAP) bacteria are heterotrophic bacteria that supply their metabolism with light energy harvested by bacteriochlorophyll-a-containing reaction centers. Despite their substantial contribution to bacterial biomass, microbial food webs, and carbon cycle, their phenology in freshwater lakes remains unknown. Hence, we investigated seasonal variations of AAP abundance and community composition biweekly across 3 years in a temperate, meso-oligotrophic freshwater lake. RESULTS: AAP bacteria displayed a clear seasonal trend with a spring maximum following the bloom of phytoplankton and a secondary maximum in autumn. As the AAP bacteria represent a highly diverse assemblage of species, we followed their seasonal succession using the amplicon sequencing of the pufM marker gene. To enhance the accuracy of the taxonomic assignment, we developed new pufM primers that generate longer amplicons and compiled the currently largest database of pufM genes, comprising 3633 reference sequences spanning all phyla known to contain AAP species. With this novel resource, we demonstrated that the majority of the species appeared during specific phases of the seasonal cycle, with less than 2% of AAP species detected during the whole year. AAP community presented an indigenous freshwater nature characterized by high resilience and heterogenic adaptations to varying conditions of the freshwater environment. CONCLUSIONS: Our findings highlight the substantial contribution of AAP bacteria to the carbon flow and ecological dynamics of lakes and unveil a recurrent and dynamic seasonal succession of the AAP community. By integrating this information with the indicator of primary production (Chlorophyll-a) and existing ecological models, we show that AAP bacteria play a pivotal role in the recycling of dissolved organic matter released during spring phytoplankton bloom. We suggest a potential role of AAP bacteria within the context of the PEG model and their consideration in further ecological models.

Flexible genomic island conservation across freshwater and marine Methylophilaceae.

The evolutionary trajectory of Methylophilaceae includes habitat transitions from freshwater sediments to freshwater and marine pelagial that resulted in genome reduction (genome-streamlining) of the pelagic taxa. However, the extent of genetic similarities in the genomic structure and microdiversity of the two genome-streamlined pelagic lineages (freshwater "Ca. Methylopumilus" and the marine OM43 lineage) has so far never been compared. Here, we analyzed complete genomes of 91 "Ca. Methylopumilus" strains isolated from 14 lakes in Central Europe and 12 coastal marine OM43 strains. The two lineages showed a remarkable niche differentiation with clear species-specific differences in habitat preference and seasonal distribution. On the other hand, we observed a synteny preservation in their genomes by having similar locations and types of flexible genomic islands (fGIs). Three main fGIs were identified: a replacement fGI acting as phage defense, an additive fGI harboring metabolic and resistance-related functions, and a tycheposon containing nitrogen-, thiamine-, and heme-related functions. The fGIs differed in relative abundances in metagenomic datasets suggesting different levels of variability ranging from strain-specific to population-level adaptations. Moreover, variations in one gene seemed to be responsible for different growth at low substrate concentrations and a potential biogeographic separation within one species. Our study provides a first insight into genomic microdiversity of closely related taxa within the family Methylophilaceae and revealed remarkably similar dynamics involving mobile genetic elements and recombination between freshwater and marine family members.

Isolation of a widespread giant virus implicated in cryptophyte bloom collapse.

Photosynthetic cryptophytes are ubiquitous protists that are major participants in the freshwater phytoplankton bloom at the onset of spring. Mortality due to change in environmental conditions and grazing have been recognized as key factors contributing to bloom collapse. In contrast, the role of viral outbreaks as factors terminating phytoplankton blooms remains unknown from freshwaters. Here, we isolated and characterized a cryptophyte virus contributing to the annual collapse of a natural cryptophyte spring bloom population. This viral isolate is also representative for a clade of abundant giant viruses (phylum Nucleocytoviricota) found in freshwaters all over the world.

Isolation of phages infecting the abundant freshwater Actinobacteriota order 'Ca. Nanopelagicales'.

Low-GC Actinobacteriota of the order 'Ca. Nanopelagicales' (also known as acI or hgcI clade) are abundant in freshwaters around the globe. Extensive predation pressure by phages has been assumed to be the reason for their high levels of microdiversity. So far, however, only a few metagenome-assembled phages have been proposed to infect them and no phages have been isolated. Taking advantage of recent advances in the cultivation of 'Ca. Nanopelagicales' we isolated a novel species of its genus 'Ca. Planktophila'. Using this isolate as bait, we cultivated the first two phages infecting this abundant bacterial order. Both genomes contained a whiB-like transcription factor and a RNA polymerase sigma-70 factor, which might aid in manipulating their host's metabolism. Both phages encoded a glycosyltransferase and one an anti-restriction protein, potential means to evade degradation of their DNA by nucleases present in the host genome. The two phage genomes shared only 6% of their genome with their closest relatives, with whom they form a previously uncultured family of actinophages within the Caudoviricetes. Read recruitment analyses against globally distributed metagenomes revealed the endemic distribution of this group of phages infecting 'Ca. Nanopelagicales'. The recruitment pattern against metagenomes from the isolation site and the modular distribution of shared genes between the two phages indicate high levels of horizontal gene transfer, likely mirroring the microdiversity of their host in the evolutionary arms race between host and phage.

Postglacial adaptations enabled colonization and quasi-clonal dispersal of ammonia-oxidizing archaea in modern European large lakes.

Ammonia-oxidizing archaea (AOA) play a key role in the aquatic nitrogen cycle. Their genetic diversity is viewed as the outcome of evolutionary processes that shaped ancestral transition from terrestrial to marine habitats. However, current genome-wide insights into AOA evolution rarely consider brackish and freshwater representatives or provide their divergence timeline in lacustrine systems. An unbiased global assessment of lacustrine AOA diversity is critical for understanding their origins, dispersal mechanisms, and ecosystem roles. Here, we leveraged continental-scale metagenomics to document that AOA species diversity in freshwater systems is remarkably low compared to marine environments. We show that the uncultured freshwater AOA, "Candidatus Nitrosopumilus limneticus," is ubiquitous and genotypically static in various large European lakes where it evolved 13 million years ago. We find that extensive proteome remodeling was a key innovation for freshwater colonization of AOA. These findings reveal the genetic diversity and adaptive mechanisms of a keystone species that has survived clonally in lakes for millennia.

High-resolution metagenomic reconstruction of the freshwater spring bloom.

BACKGROUND: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. RESULTS: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. CONCLUSION: We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom. Video Abstract.

Cryptic and ubiquitous aplastidic cryptophytes are key freshwater flagellated bacterivores.

Morphology-based microscopic approaches are insufficient for a taxonomic classification of bacterivorous heterotrophic nanoflagellates (HNF) in aquatic environments since their cells do not display reliably distinguishable morphological features. This leads to a considerable lack of ecological insights into this large and taxonomically diverse functional guild. Here, we present a combination of fluorescence in situ hybridization followed by catalyzed reporter deposition (CARD-FISH) and environmental sequence analyses which revealed that morphologically indistinguishable, so far largely cryptic and uncultured aplastidic cryptophytes are ubiquitous and prominent protistan bacterivores in diverse freshwater ecosystems. Using a general probe for Cryptophyceae and its heterotrophic CRY1 lineage, we analyzed different water layers in 24 freshwater lakes spanning a broad range of trophic states, sizes and geographical locations. We show that bacterivorous aplastidic cryptophytes and the CRY1 lineage accounted for ca. 2/3 and » of total HNF, respectively, in both epilimnetic and hypolimnetic samples. These heterotrophic cryptophytes were generally smaller and more abundant than their chloroplast-bearing counterparts. They had high uptake rates of bacteria, hinting at their important roles in channeling carbon flow from prokaryotes to higher trophic levels. The worldwide ubiquity of Cryptophyceae and its CRY1 lineage was supported by 18S rRNA gene sequence analyses across a diverse set of 297 freshwater metagenomes. While cryptophytes have been considered to be mainly plastidic "algae", we show that it is the aplastidic counterparts that contribute considerably to bacterial mortality rates. Additionally, our results suggest an undiscovered diversity hidden amongst these abundant and morphologically diverse aplastidic cryptophytes.

The Evolutionary Kaleidoscope of Rhodopsins.

Rhodopsins are widely distributed across all domains of life where they perform a plethora of functions through the conversion of electromagnetic radiation into physicochemical signals. As a result of an extensive survey of available genomic and metagenomic sequencing data, we reported the existence of novel clades and exotic sequence motifs scattered throughout the evolutionary radiations of both Type-1 and Type-3 rhodopsins that will likely enlarge the optogenetics toolbox. We expanded the typical rhodopsin blueprint by showing that a highly conserved and functionally important arginine residue (i.e., Arg82) was substituted multiple times during evolution by an extensive amino acid spectrum. We proposed the umbrella term Alt-rhodopsins (AltRs) for all such proteins that departed Arg82 orthodoxy. Some AltRs formed novel clades in the rhodopsin phylogeny and were found in giant viruses. Some newly uncovered AltRs were phylogenetically close to heliorhodopsins, which allowed a closer examination of the phylogenetic border between Type-1 rhodopsins and heliorhodopsins. Comprehensive phylogenetic trees and ancestral sequence reconstructions allowed us to advance the hypothesis that proto-heliorhodopsins were a eukaryotic innovation before their subsequent diversification into the extant Type-3 rhodopsins. IMPORTANCE The rhodopsin scaffold is remarkably versatile and widespread, coupling light availability to energy production and other light-dependent cellular responses with minor alterations to critical residues. We described an unprecedented spectrum of substitutions at one of the most conserved amino acids in the rhodopsin fold, Arg82. We denoted such phylogenetically diverse rhodopsins with the umbrella name Alt-rhodopsins (AltR) and described a distinct branch of AltRs in giant viruses. Intriguingly, some AltRs were the closest phylogenetic neighbors to Heliorhodopsins (HeRs) whose origins have remained enigmatic. Our analyses of HeR origins in the light of AltRs led us to posit a most unusual evolutionary trajectory that suggested a eukaryotic origin for HeRs before their diversification in prokaryotes.

Ecogenomics sheds light on diverse lifestyle strategies in freshwater CPR.

BACKGROUND: The increased use of metagenomics and single-cell genomics led to the discovery of organisms from phyla with no cultivated representatives and proposed new microbial lineages such as the candidate phyla radiation (CPR or Patescibacteria). These bacteria have peculiar ribosomal structures, reduced metabolic capacities, small genome, and cell sizes, and a general host-associated lifestyle was proposed for the radiation. So far, most CPR genomes were obtained from groundwaters; however, their diversity, abundance, and role in surface freshwaters is largely unexplored. Here, we attempt to close these knowledge gaps by deep metagenomic sequencing of 119 samples of 17 different freshwater lakes located in Europe and Asia. Moreover, we applied Fluorescence in situ Hybridization followed by Catalyzed Reporter Deposition (CARD-FISH) for a first visualization of distinct CPR lineages in freshwater samples. RESULTS: A total of 174 dereplicated metagenome-assembled genomes (MAGs) of diverse CPR lineages were recovered from the investigated lakes, with a higher prevalence from hypolimnion samples (162 MAGs). They have reduced genomes (median size 1 Mbp) and were generally found in low abundances (0.02-14.36 coverage/Gb) and with estimated slow replication rates. The analysis of genomic traits and CARD-FISH results showed that the radiation is an eclectic group in terms of metabolic capabilities and potential lifestyles, ranging from what appear to be free-living lineages to host- or particle-associated groups. Although some complexes of the electron transport chain were present in the CPR MAGs, together with ion-pumping rhodopsins and heliorhodopsins, we believe that they most probably adopt a fermentative metabolism. Terminal oxidases might function in O2 scavenging, while heliorhodopsins could be involved in mitigation against oxidative stress. CONCLUSIONS: A high diversity of CPR MAGs was recovered, and distinct CPR lineages did not seem to be limited to lakes with specific trophic states. Their reduced metabolic capacities resemble the ones described for genomes in groundwater and animal-associated samples, apart from Gracilibacteria that possesses more complete metabolic pathways. Even though this radiation is mostly host-associated, we also observed organisms from different clades (ABY1, Paceibacteria, Saccharimonadia) that appear to be unattached to any other organisms or were associated with 'lake snow' particles (ABY1, Gracilibacteria), suggesting a broad range of potential life-strategies in this phylum. Video Abstract.

Heliorhodopsin Evolution Is Driven by Photosensory Promiscuity in Monoderms.

Rhodopsins are light-activated proteins displaying an enormous versatility of function as cation/anion pumps or sensing environmental stimuli and are widely distributed across all domains of life. Even with wide sequence divergence and uncertain evolutionary linkages between microbial (type 1) and animal (type 2) rhodopsins, the membrane orientation of the core structural scaffold of both was presumed universal. This was recently amended through the discovery of heliorhodopsins (HeRs; type 3), that, in contrast to known rhodopsins, display an inverted membrane topology and yet retain similarities in sequence, structure, and the light-activated response. While no ion-pumping activity has been demonstrated for HeRs and multiple crystal structures are available, fundamental questions regarding their cellular and ecological function or even their taxonomic distribution remain unresolved. Here, we investigated HeR function and distribution using genomic/metagenomic data with protein domain fusions, contextual genomic information, and gene coexpression analysis with strand-specific metatranscriptomics. We bring to resolution the debated monoderm/diderm occurrence patterns and show that HeRs are restricted to monoderms. Moreover, we provide compelling evidence that HeRs are a novel type of sensory rhodopsins linked to histidine kinases and other two-component system genes across phyla. In addition, we also describe two novel putative signal-transducing domains fused to some HeRs. We posit that HeRs likely function as generalized light-dependent switches involved in the mitigation of light-induced oxidative stress and metabolic circuitry regulation. Their role as sensory rhodopsins is corroborated by their photocycle dynamics and their presence/function in monoderms is likely connected to the higher sensitivity of these organisms to light-induced damage. IMPORTANCE Heliorhodopsins are enigmatic, novel rhodopsins with a membrane orientation that is opposite to all known rhodopsins. However, their cellular and ecological functions are unknown, and even their taxonomic distribution remains a subject of debate. We provide evidence that HeRs are a novel type of sensory rhodopsins linked to histidine kinases and other two-component system genes across phyla boundaries. In support of this, we also identify two novel putative signal transducing domains in HeRs that are fused with them. We also observe linkages of HeRs to genes involved in mitigation of light-induced oxidative stress and increased carbon and nitrogen metabolism. Finally, we synthesize these findings into a framework that connects HeRs with the cellular response to light in monoderms, activating light-induced oxidative stress defenses along with carbon/nitrogen metabolic circuitries. These findings are consistent with the evolutionary, taxonomic, structural, and genomic data available so far.

Quorum-Sensing Signals from Epibiont Mediate the Induction of Novel Microviridins in the Mat-Forming Cyanobacterial Genus Nostoc.

The regulation of the production of oligopeptides is essential in understanding their ecological role in complex microbial communities, including harmful cyanobacterial blooms. The role of chemical communication between the cyanobacterium and the microbial community harbored as epibionts within its phycosphere is at an initial stage of research, and little is understood about its specificity. Here, we present insight into the role of a bacterial epibiont in regulating the production of novel microviridins isolated from Nostoc, an ecologically important cyanobacterial genus. Microviridins are well-known elastase inhibitors with presumed antigrazing effects. Heterologous expression and identification of specific signal molecules from the epibiont suggest the role of a quorum-sensing-based interaction. Furthermore, physiological experiments show an increase in microviridin production without affecting cyanobacterial growth and photosynthetic activity. Simultaneously, oligopeptides presenting a selective inhibition pattern provide support for their specific function in response to the presence of cohabitant epibionts. Thus, the chemical interaction revealed in our study provides an example of an interspecies signaling pathway monitoring the bacterial flora around the cyanobacterial filaments and the induction of intrinsic species-specific metabolic responses. IMPORTANCE The regulation of the production of cyanopeptides beyond microcystin is essential to understand their ecological role in complex microbial communities, e.g., harmful cyanobacterial blooms. The role of chemical communication between the cyanobacterium and the epibionts within its phycosphere is at an initial stage of research, and little is understood about its specificity. The frequency of cyanopeptide occurrence also demonstrates the need to understand the contribution of cyanobacterial peptides to the overall biological impact of cyanopeptides on aquatic organisms and vertebrates, including humans. Our results shed light on the epibiont control of microviridin production via quorum-sensing mechanisms, and we posit that such mechanisms may be widespread in natural cyanobacterial bloom community regulation.

Common Presence of Phototrophic Gemmatimonadota in Temperate Freshwater Lakes.

Members of the bacterial phylum Gemmatimonadota are ubiquitous in most natural environments and represent one of the top 10 most abundant bacterial phyla in soil. Sequences affiliated with Gemmatimonadota were also reported from diverse aquatic habitats; however, it remains unknown whether they are native organisms or represent bacteria passively transported from sediment or soil. To address this question, we analyzed metagenomes constructed from five freshwater lakes in central Europe. Based on the 16S rRNA gene frequency, Gemmatimonadota represented from 0.02 to 0.6% of all bacteria in the epilimnion and between 0.1 and 1% in the hypolimnion. These proportions were independently confirmed using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Some cells in the epilimnion were attached to diatoms (Fragilaria sp.) or cyanobacteria (Microcystis sp.), which suggests a close association with phytoplankton. In addition, we reconstructed 45 metagenome-assembled genomes (MAGs) related to Gemmatimonadota They represent several novel lineages, which persist in the studied lakes during the seasons. Three lineages contained photosynthesis gene clusters. One of these lineages was related to Gemmatimonas phototrophica and represented the majority of Gemmatimonadota retrieved from the lakes' epilimnion. The other two lineages came from hypolimnion and probably represented novel photoheterotrophic genera. None of these phototrophic MAGs contained genes for carbon fixation. Since most of the identified MAGs were present during the whole year and cells associated with phytoplankton were observed, we conclude that they represent truly limnic Gemmatimonadota distinct from the previously described species isolated from soils or sediments.IMPORTANCE Photoheterotrophic bacterial phyla such as Gemmatimonadota are key components of many natural environments. Its first photoheterotrophic cultured member, Gemmatimonas phototrophica, was isolated in 2014 from a shallow lake in the Gobi Desert. It contains a unique type of photosynthetic complex encoded by a set of genes which were likely received via horizontal transfer from Proteobacteria We were intrigued to discover how widespread this group is in the natural environment. In the presented study, we analyzed 45 metagenome-assembled genomes (MAGs) that were obtained from five freshwater lakes in Switzerland and Czechia. Interestingly, it was found that phototrophic Gemmatimonadota are relatively common in euphotic zones of the studied lakes, whereas heterotrophic Gemmatimonadota prevail in deeper waters. Moreover, our analysis of the MAGs documented that these freshwater species contain almost the same set of photosynthesis genes identified before in Gemmatimonas phototrophica originating from the Gobi Desert.

Culturomics-based genomics sheds light on the ecology of the new haloarchaeal genus Halosegnis.

The development of culture-independent techniques has revolutionized our understanding of microbial ecology, especially through the illustration of the vast gap between the environmentally abundant microbial diversity and that accessible through cultivation. However, culture-based approaches are not only crucial for understanding the evolutionary, metabolic and ecological milieu of microbial diversity but also for the development of novel biotechnological applications. In this study, we used a culturomics-based approach in order to isolate novel microbial taxa from hypersaline environments (i.e. Isla Cristina and Isla Bacuta salterns in Huelva, Spain). We managed to obtain axenic cultures of four haloarchaeal strains that belong to a new haloarchaeal genus and to obtain their genomic sequences. The phylogenomic and phylogenetic analyses (together with AAI, ANI and digital DDH indices) showed that the isolates constitute two new species, for which we propose the names Halosegnis longus sp. nov. and Halosegnis rubeus sp. nov. The genomic-based metabolic reconstructions indicated that members of this new haloarchaeal genus have photoheterotrophic aerobic lifestyle with a typical salt-in signature. 16S rRNA gene sequence reads abundance profiles and genomic recruitment analyses revealed that the Halosegnis genus has a worldwide geographical distribution, reaching high abundance (up to 8%) in habitats with intermediate salinities.

Expanded Diversity and Metabolic Versatility of Marine Nitrite-Oxidizing Bacteria Revealed by Cultivation- and Genomics-Based Approaches.

Nitrite-oxidizing bacteria (NOB) are ubiquitous and abundant microorganisms that play key roles in global nitrogen and carbon biogeochemical cycling. Despite recent advances in understanding NOB physiology and taxonomy, currently very few cultured NOB or representative NOB genome sequences from marine environments exist. In this study, we employed enrichment culturing and genomic approaches to shed light on the phylogeny and metabolic capacity of marine NOB. We successfully enriched two marine NOB (designated MSP and DJ) and obtained a high-quality metagenome-assembled genome (MAG) from each organism. The maximum nitrite oxidation rates of the MSP and DJ enrichment cultures were 13.8 and 30.0 µM nitrite per day, respectively, with these optimum rates occurring at 0.1 mM and 0.3 mM nitrite, respectively. Each enrichment culture exhibited a different tolerance to various nitrite and salt concentrations. Based on phylogenomic position and overall genome relatedness indices, both NOB MAGs were proposed as novel taxa within the Nitrospinota and Nitrospirota phyla. Functional predictions indicated that both NOB MAGs shared many highly conserved metabolic features with other NOB. Both NOB MAGs encoded proteins for hydrogen and organic compound metabolism and defense mechanisms for oxidative stress. Additionally, these organisms may have the genetic potential to produce cobalamin (an essential enzyme cofactor that is limiting in many environments) and, thus, may play an important role in recycling cobalamin in marine sediment. Overall, this study appreciably expands our understanding of the Nitrospinota and Nitrospirota phyla and suggests that these NOB play important biogeochemical roles in marine habitats.IMPORTANCE Nitrification is a key process in the biogeochemical and global nitrogen cycle. Nitrite-oxidizing bacteria (NOB) perform the second step of aerobic nitrification (converting nitrite to nitrate), which is critical for transferring nitrogen to other organisms for assimilation or energy. Despite their ecological importance, there are few cultured or genomic representatives from marine systems. Here, we obtained two NOB (designated MSP and DJ) enriched from marine sediments and estimated the physiological and genomic traits of these marine microbes. Both NOB enrichment cultures exhibit distinct responses to various nitrite and salt concentrations. Genomic analyses suggest that these NOB are metabolically flexible (similar to other previously described NOB) yet also have individual genomic differences that likely support distinct niche distribution. In conclusion, this study provides more insights into the ecological roles of NOB in marine environments.

A freshwater radiation of diplonemids.

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Japanese deep freshwater lakes. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and catalysed reporter deposition-fluorescent in situ hybridization (CARD-FISH). We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to a marine clade, which are mainly isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single-cell genomes of marine diplonemids show they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone.

Visualization of Lokiarchaeia and Heimdallarchaeia (Asgardarchaeota) by Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition (CARD-FISH).

Metagenome-assembled genomes (MAGs) of Asgardarchaeota have been recovered from a variety of habitats, broadening their environmental distribution and providing access to the genetic makeup of this archaeal lineage. The recent success in cultivating the first representative of Lokiarchaeia was a breakthrough in science at large and gave rise to new hypotheses about the evolution of eukaryotes. Despite their singular phylogenetic position at the base of the eukaryotic tree of life, the morphology of these bewildering organisms remains a mystery, except for the report of an unusual morphology with long, branching protrusions of the cultivated Lokiarchaeion strain "Candidatus Prometheoarchaeum syntrophicum" MK-D1. In order to visualize this elusive group, we applied a combination of fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed CARD-FISH probes for Heimdallarchaeia and Lokiarchaeia lineages, and provide the first visual evidence for Heimdallarchaeia and new images of a lineage of Lokiarchaeia that is different from the cultured representative. Here, we show that while Heimdallarchaeia are characterized by a uniform cellular morphology typified by a centralized DNA localization, Lokiarchaeia display a plethora of shapes and sizes that likely reflect their broad phylogenetic diversity and ecological distribution.IMPORTANCE Asgardarchaeota are considered to be the closest relatives to modern eukaryotes. These enigmatic microbes have been mainly studied using metagenome-assembled genomes (MAGs). Only very recently, a first member of Lokiarchaeia was isolated and characterized in detail; it featured a striking morphology with long, branching protrusions. In order to visualize additional members of the phylum Asgardarchaeota, we applied a fluorescence in situ hybridization technique and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed probes for Heimdallarchaeia and Lokiarchaeia lineages. We provide the first visual evidence for Heimdallarchaeia that are characterized by a uniform cellular morphology typified by an apparently centralized DNA localization. Further, we provide new images of a lineage of Lokiarchaeia that is different from the cultured representative and with multiple morphologies, ranging from small ovoid cells to long filaments. This diversity in observed cell shapes is likely owing to the large phylogenetic diversity within Asgardarchaeota, the vast majority of which remain uncultured.

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps.

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

Correction to: Phage-centric ecological interactions in aquatic ecosystems revealed through ultra-deep metagenomics.

Following publication of the original article [1], the authors reported that an affiliation of the first author was missing.

Phage-centric ecological interactions in aquatic ecosystems revealed through ultra-deep metagenomics.

The persistent inertia in the ability to culture environmentally abundant microbes from aquatic ecosystems represents an obstacle in disentangling the complex web of ecological interactions spun by a diverse assortment of participants (pro- and eukaryotes and their viruses). In aquatic microbial communities, the numerically most abundant actors, the viruses, remain the most elusive, and especially in freshwaters their identities and ecology remain unknown. Here, using ultra-deep metagenomic sequencing from pelagic freshwater habitats, we recovered complete genomes of > 2000 phages, including small "miniphages" and large "megaphages" infecting iconic freshwater prokaryotic lineages. For instance, abundant freshwater Actinobacteria support infection by a very broad size range of phages (13-200 Kb). We describe many phages encoding genes that likely afford protection to their host from reactive oxygen species (ROS) in the aquatic environment and in the oxidative burst in protist phagolysosomes (phage-mediated ROS defense). Spatiotemporal abundance analyses of phage genomes revealed evanescence as the primary dynamic in upper water layers, where they displayed short-lived existences. In contrast, persistence was characteristic for the deeper layers where many identical phage genomes were recovered repeatedly. Phage and host abundances corresponded closely, with distinct populations displaying preferential distributions in different seasons and depths, closely mimicking overall stratification and mixis.

New Halonotius Species Provide Genomics-Based Insights Into Cobalamin Synthesis in Haloarchaea.

Hypersaline aquatic and terrestrial ecosystems display a cosmopolitan distribution. These environments teem with microbes and harbor a plethora of prokaryotic lineages that evaded ecological characterization due to the prior inability to cultivate them or to access their genomic information. In order to close the current knowledge gap, we performed two sampling and isolation campaigns in the saline soils of the Odiel Saltmarshes and the salterns of Isla Cristina (Huelva, Spain). From the isolated haloarchaeal strains subjected to high-throughput phylogenetic screening, two were chosen (F15BT and F9-27T) for physiological and genomic characterization due of their relatedness to the genus Halonotius. Comparative genomic analyses were carried out between the isolated strains and the genomes of previously described species Halonotius pteroides CECT 7525T, Halonotius aquaticus F13-13T and environmentaly recovered metagenome-assembled representatives of the genus Halonotius. The topology of the phylogenomic tree showed agreement with the phylogenetic ones based on 16S rRNA and rpoB' genes, and together with average amino acid and nucleotide identities suggested the two strains as novel species within the genus. We propose the names Halonotius terrestris sp. nov. (type strain F15BT = CECT 9688T = CCM 8954T) and Halonotius roseus sp. nov. (type strain F9-27T = CECT 9745T = CCM 8956T) for these strains. Comparative genomic analyses within the genus highlighted a typical salt-in signature, characterized by acidic proteomes with low isoelectric points, and indicated heterotrophic aerobic lifestyles. Genome-scale metabolic reconstructions revealed that the newly proposed species encode all the necessary enzymatic reactions involved in cobalamin (vitamin B12) biosynthesis. Based on the worldwide distribution of the genus and its abundance in hypersaline habitats we postulate that its members perform a critical function by being able to provide "expensive" commodities (i.e., vitamin B12) to the halophilic microbial communities at large.