Marine viruses disperse bidirectionally along the natural water cycle.

Marine viruses in seawater have frequently been studied, yet their dispersal from neuston ecosystems at the air-sea interface towards the atmosphere remains a knowledge gap. Here, we show that 6.2% of the studied virus population were shared between air-sea interface ecosystems and rainwater. Virus enrichment in the 1-mm thin surface microlayer and sea foams happened selectively, and variant analysis proved virus transfer to aerosols collected at ~2 m height above sea level and rain. Viruses detected in rain and these aerosols showed a significantly higher percent G/C base content compared to marine viruses. CRISPR spacer matches of marine prokaryotes to foreign viruses from rainwater prove regular virus-host encounters at the air-sea interface. Our findings on aerosolization, adaptations, and dispersal support transmission of viruses along the natural water cycle.

Ecogenomics and cultivation reveal distinctive viral-bacterial communities in the surface microlayer of a Baltic Sea slick.

Visible surface films, termed slicks, can extensively cover freshwater and marine ecosystems, with coastal regions being particularly susceptible to their presence. The sea-surface microlayer (SML), the upper 1-mm at the air-water interface in slicks (herein slick SML) harbors a distinctive bacterial community, but generally little is known about SML viruses. Using flow cytometry, metagenomics, and cultivation, we characterized viruses and bacteria in a brackish slick SML in comparison to non-slick SML as well as seawater below slick and non-slick areas (subsurface water = SSW). Size-fractionated filtration of all samples distinguished viral attachment to hosts and particles. The slick SML contained higher abundances of virus-like particles, prokaryotic cells, and dissolved organic carbon compared to non-slick SML and SSW. The community of 428 viral operational taxonomic units (vOTUs), 426 predicted as lytic, distinctly differed across all size fractions in the slick SML compared to non-slick SML and SSW. Specific metabolic profiles of bacterial metagenome-assembled genomes and isolates in the slick SML included a prevalence of genes encoding motility and carbohydrate-active enzymes (CAZymes). Several vOTUs were enriched in slick SML, and many virus variants were associated with particles. Nine vOTUs were only found in slick SML, six of them being targeted by slick SML-specific clustered-regularly interspaced short palindromic repeats (CRISPR) spacers likely originating from Gammaproteobacteria. Moreover, isolation of three previously unknown lytic phages for Alishewanella sp. and Pseudoalteromonas tunicata, abundant and actively replicating slick SML bacteria, suggests that viral activity in slicks contributes to biogeochemical cycling in coastal ecosystems.

A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.

Spatio-functional organization in virocells of small uncultivated archaea from the deep biosphere.

Despite important ecological roles posited for virocells (i.e., cells infected with viruses), studying individual cells in situ is technically challenging. We introduce here a novel correlative microscopic approach to study the ecophysiology of virocells. By conducting concerted virusFISH, 16S rRNA FISH, and scanning electron microscopy interrogations of uncultivated archaea, we linked morphologies of various altiarchaeal cells to corresponding phylogenetic signals and indigenous virus infections. While uninfected cells exhibited moderate separation between fluorescence signals of ribosomes and DNA, virocells displayed complete cellular segregation of chromosomal DNA from viral DNA, the latter co-localizing with host ribosome signals. A similar spatial separation was observed in dividing cells, with viral signals congregating near ribosomes at the septum. These observations suggest that replication of these uncultivated viruses occurs alongside host ribosomes, which are used to generate the required proteins for virion assembly. Heavily infected cells sometimes displayed virus-like particles attached to their surface, which agree with virus structures in cells observed via transmission electron microscopy. Consequently, this approach is the first to link genomes of uncultivated viruses to their respective structures and host cells. Our findings shed new light on the complex ecophysiology of archaeal virocells in deep subsurface biofilms and provide a solid framework for future in situ studies of virocells.

Seasonality and Strain Specificity Drive Rapid Co-evolution in an Ostreococcus-Virus System from the Western Baltic Sea.

Marine viruses are a major driver of phytoplankton mortality and thereby influence biogeochemical cycling of carbon and other nutrients. Phytoplankton-targeting viruses are important components of ecosystem dynamics, but broad-scale experimental investigations of host-virus interactions remain scarce. Here, we investigated in detail a picophytoplankton (size 1 µm) host's responses to infections by species-specific viruses from distinct geographical regions and different sampling seasons. Specifically, we used Ostreococcus tauri and O. mediterraneus and their viruses (size ca. 100 nm). Ostreococcus sp. is globally distributed and, like other picoplankton species, play an important role in coastal ecosystems at certain times of the year. Further, Ostreococcus sp. is a model organism, and the Ostreococcus-virus system is well-known in marine biology. However, only few studies have researched its evolutionary biology and the implications thereof for ecosystem dynamics. The Ostreococcus strains used here stem from different regions of the Southwestern Baltic Sea that vary in salinity and temperature and were obtained during several cruises spanning different sampling seasons. Using an experimental cross-infection set-up, we explicitly confirm species and strain specificity in Ostreococcus sp. from the Baltic Sea. Moreover, we found that the timing of virus-host co-existence was a driver of infection patterns as well. In combination, these findings prove that host-virus co-evolution can be rapid in natural systems.

Virus-Host Dynamics in Archaeal Groundwater Biofilms and the Associated Bacterial Community Composition.

Spatial and temporal distribution of lytic viruses in deep groundwater remains unexplored so far. Here, we tackle this gap of knowledge by studying viral infections of Altivir_1_MSI in biofilms dominated by the uncultivated host Candidatus Altiarchaeum hamiconexum sampled from deep anoxic groundwater over a period of four years. Using virus-targeted direct-geneFISH (virusFISH) whose detection efficiency for individual viral particles was 15%, we show a significant and steady increase of virus infections from 2019 to 2022. Based on fluorescence micrographs of individual biofilm flocks, we determined different stages of viral infections in biofilms for single sampling events, demonstrating the progression of infection of biofilms in deep groundwater. Biofilms associated with many host cells undergoing lysis showed a substantial accumulation of filamentous microbes around infected cells probably feeding off host cell debris. Using 16S rRNA gene sequencing across ten individual biofilm flocks from one sampling event, we determined that the associated bacterial community remains relatively constant and was dominated by sulfate-reducing members affiliated with Desulfobacterota. Given the stability of the virus-host interaction in these deep groundwater samples, we postulate that the uncultivated virus-host system described herein represents a suitable model system for studying deep biosphere virus-host interactions in future research endeavors.

Women in the European Virus Bioinformatics Center.

Viruses are the cause of a considerable burden to human, animal and plant health, while on the other hand playing an important role in regulating entire ecosystems. The power of new sequencing technologies combined with new tools for processing "Big Data" offers unprecedented opportunities to answer fundamental questions in virology. Virologists have an urgent need for virus-specific bioinformatics tools. These developments have led to the formation of the European Virus Bioinformatics Center, a network of experts in virology and bioinformatics who are joining forces to enable extensive exchange and collaboration between these research areas. The EVBC strives to provide talented researchers with a supportive environment free of gender bias, but the gender gap in science, especially in math-intensive fields such as computer science, persists. To bring more talented women into research and keep them there, we need to highlight role models to spark their interest, and we need to ensure that female scientists are not kept at lower levels but are given the opportunity to lead the field. Here we showcase the work of the EVBC and highlight the achievements of some outstanding women experts in virology and viral bioinformatics.

Host-Associated Phages Disperse across the Extraterrestrial Analogue Antarctica.

Extreme Antarctic conditions provide one of the closest analogues of extraterrestrial environments. Since air and snow samples, especially from polar regions, yield DNA amounts in the lower picogram range, binning of prokaryotic genomes is challenging and renders studying the dispersal of biological entities across these environments difficult. Here, we hypothesized that dispersal of host-associated bacteriophages (adsorbed, replicating, or prophages) across the Antarctic continent can be tracked via their genetic signatures, aiding our understanding of virus and host dispersal across long distances. Phage genome fragments (PGFs) reconstructed from surface snow metagenomes of three Antarctic stations were assigned to four host genomes, mainly Betaproteobacteria, including Ralstonia spp. We reconstructed the complete genome of a temperate phage with nearly complete alignment to a prophage in the reference genome of Ralstonia pickettii 12D. PGFs from different stations were related to each other at the genus level and matched similar hosts. Metagenomic read mapping and nucleotide polymorphism analysis revealed a wide dispersal of highly identical PGFs, 13 of which were detected in seawater from the Western Antarctic Peninsula at a distance of 5,338 km from the snow sampling stations. Our results suggest that host-associated phages, especially of Ralstonia sp., disperse over long distances despite the harsh conditions of the Antarctic continent. Given that 14 phages associated with two R. pickettii draft genomes isolated from space equipment were identified, we conclude that Ralstonia phages are ideal mobile genetic elements to track dispersal and contamination in ecosystems relevant for astrobiology. IMPORTANCE Host-associated phages of the bacterium Ralstonia identified in snow samples can be used to track microbial dispersal over thousands of kilometers across the Antarctic continent, which functions as an extraterrestrial analogue because of its harsh environmental conditions. Due to the presence of these bacteria carrying genome-integrated prophages on space-related equipment and the potential for dispersal of host-associated phages demonstrated here, our work has implications for planetary protection, a discipline in astrobiology interested in preventing contamination of celestial bodies with alien biomolecules or forms of life.

Genetic diversity in terrestrial subsurface ecosystems impacted by geological degassing.

Earth's mantle releases 38.7 ± 2.9 Tg/yr CO2 along with other reduced and oxidized gases to the atmosphere shaping microbial metabolism at volcanic sites across the globe, yet little is known about its impact on microbial life under non-thermal conditions. Here, we perform comparative metagenomics coupled to geochemical measurements of deep subsurface fluids from a cold-water geyser driven by mantle degassing. Key organisms belonging to uncultivated Candidatus Altiarchaeum show a global biogeographic pattern and site-specific adaptations shaped by gene loss and inter-kingdom horizontal gene transfer. Comparison of the geyser community to 16 other publicly available deep subsurface sites demonstrate a conservation of chemolithoautotrophic metabolism across sites. In silico replication measures suggest a linear relationship of bacterial replication with ecosystems depth with the exception of impacted sites, which show near surface characteristics. Our results suggest that subsurface ecosystems affected by geological degassing are hotspots for microbial life in the deep biosphere.

Virus-associated organosulfur metabolism in human and environmental systems.

Viruses influence the fate of nutrients and human health by killing microorganisms and altering metabolic processes. Organosulfur metabolism and biologically derived hydrogen sulfide play dynamic roles in manifestation of diseases, infrastructure degradation, and essential biological processes. Although microbial organosulfur metabolism is well studied, the role of viruses in organosulfur metabolism is unknown. Here, we report the discovery of 39 gene families involved in organosulfur metabolism encoded by 3,749 viruses from diverse ecosystems, including human microbiomes. The viruses infect organisms from all three domains of life. Six gene families encode for enzymes that degrade organosulfur compounds into sulfide, whereas others manipulate organosulfur compounds and may influence sulfide production. We show that viral metabolic genes encode key enzymatic domains, are translated into protein, and are maintained after recombination, and sulfide provides a fitness advantage to viruses. Our results reveal viruses as drivers of organosulfur metabolism with important implications for human and environmental health.

Lytic archaeal viruses infect abundant primary producers in Earth's crust.

The continental subsurface houses a major portion of life's abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we use a combination of metagenomics and virus-targeted direct-geneFISH (virusFISH) to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches display resistances of Ca. Altiarchaea against eight predicted viral clades, which show genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tag and image a putatively viral genome rich in protospacers using fluorescence microscopy. VirusFISH reveals a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.

Diverse Viruses Carrying Genes for Microbial Extremotolerance in the Atacama Desert Hyperarid Soil.

Viruses play an essential role in shaping microbial community structures and serve as reservoirs for genetic diversity in many ecosystems. In hyperarid desert environments, where life itself becomes scarce and loses diversity, the interactions between viruses and host populations have remained elusive. Here, we resolved host-virus interactions in the soil metagenomes of the Atacama Desert hyperarid core, one of the harshest terrestrial environments on Earth. We show evidence of diverse viruses infecting a wide range of hosts found in sites up to 205 km apart. Viral genomes carried putative extremotolerance features (i.e., spore formation proteins) and auxiliary metabolic genes, indicating that viruses could mediate the spread of microbial resilience against environmental stress across the desert. We propose a mutualistic model of host-virus interactions in the hyperarid core where viruses seek protection in microbial cells as lysogens or pseudolysogens, while viral extremotolerance genes aid survival of their hosts. Our results suggest that the host-virus interactions in the Atacama Desert soils are dynamic and complex, shaping uniquely adapted microbiomes in this highly selective and hostile environment.IMPORTANCE Deserts are one of the largest and rapidly expanding terrestrial ecosystems characterized by low biodiversity and biomass. The hyperarid core of the Atacama Desert, previously thought to be devoid of life, is one of the harshest environments, supporting only scant biomass of highly adapted microbes. While there is growing evidence that viruses play essential roles in shaping the diversity and structure of nearly every ecosystem, very little is known about the role of viruses in desert soils, especially where viral contact with viable hosts is significantly reduced. Our results demonstrate that diverse viruses are widely dispersed across the desert, potentially spreading key stress resilience and metabolic genes to ensure host survival. The desertification accelerated by climate change expands both the ecosystem cover and the ecological significance of the desert virome. This study sheds light on the complex virus-host interplay that shapes the unique microbiome in desert soils.

Sea foams are ephemeral hotspots for distinctive bacterial communities contrasting sea-surface microlayer and underlying surface water.

The occurrence of foams at oceans' surfaces is patchy and generally short-lived, but a detailed understanding of bacterial communities inhabiting sea foams is lacking. Here, we investigated how marine foams differ from the sea-surface microlayer (SML), a <1-mm-thick layer at the air-sea interface, and underlying water from 1 m depth. Samples of sea foams, SML and underlying water collected from the North Sea and Timor Sea indicated that foams were often characterized by a high abundance of small eukaryotic phototrophic and prokaryotic cells as well as a high concentration of surface-active substances (SAS). Amplicon sequencing of 16S rRNA (gene) revealed distinctive foam bacterial communities compared with SML and underlying water, with high abundance of Gammaproteobacteria. Typical SML dwellers such as Pseudoalteromonas and Vibrio were highly abundant, active foam inhabitants and thus might enhance foam formation and stability by producing SAS. Despite a clear difference in the overall bacterial community composition between foam and SML, the presence of SML bacteria in foams supports the previous assumption that foam is strongly influenced by the SML. We conclude that active and abundant bacteria from interfacial habitats potentially contribute to foam formation and stability, carbon cycling and air-sea exchange processes in the ocean.

The Virioneuston: A Review on Viral⁻Bacterial Associations at Air⁻Water Interfaces.

Vast biofilm-like habitats at air⁻water interfaces of marine and freshwater ecosystems harbor surface-dwelling microorganisms, which are commonly referred to as neuston. Viruses in the microlayer, i.e., the virioneuston, remain the most enigmatic biological entities in boundary surface layers due to their potential ecological impact on the microbial loop and major air⁻water exchange processes. To provide a broad picture of the viral⁻bacterial dynamics in surface microlayers, this review compiles insights on the challenges that viruses likely encounter at air⁻water interfaces. By considering viral abundance and morphology in surface microlayers, as well as dispersal and infection mechanisms as inferred from the relevant literature, this work highlights why studying the virioneuston in addition to the bacterioneuston is a worthwhile task. In this regard, major knowledge gaps and possible future research directions are discussed.

Blue pigmentation of neustonic copepods benefits exploitation of a prey-rich niche at the air-sea boundary.

The sea-surface microlayer (SML) at the air-sea interface is a distinct, under-studied habitat compared to the subsurface and copepods, important components of ocean food webs, have developed key adaptations to exploit this niche. By using automated SML sampling, high-throughput sequencing and unmanned aerial vehicles, we report on the distribution and abundance of pontellid copepods in relation to the unique biophysicochemical signature of the SML. We found copepods in the SML even during high exposure to sun-derived ultraviolet radiation and their abundance was significantly correlated to increased algal biomass. We additionally investigated the significance of the pontellids' blue pigmentation and found that the reflectance peak of the blue pigment matched the water-leaving spectral radiance of the ocean surface. This feature could reduce high visibility at the air-sea boundary and potentially provide camouflage of copepods from their predators.

High wind speeds prevent formation of a distinct bacterioneuston community in the sea-surface microlayer.

The sea-surface microlayer (SML) at the boundary between atmosphere and hydrosphere represents a demanding habitat for bacteria. Wind speed is a crucial but poorly studied factor for its physical integrity. Increasing atmospheric burden of CO2, as suggested for future climate scenarios, may particularly act on this habitat at the air-sea interface. We investigated the effect of increasing wind speeds and different pCO2 levels on SML microbial communities in a wind-wave tunnel, which offered the advantage of low spatial and temporal variability. We found that enrichment of bacteria in the SML occurred solely at a U10 wind speed of ≤5.6 m s-1 in the tunnel and ≤4.1 m s-1 in the Baltic Sea. High pCO2 levels further intensified the bacterial enrichment in the SML during low wind speed. In addition, low wind speed and pCO2 induced the formation of a distinctive bacterial community as revealed by 16S rRNA gene fingerprints and influenced the presence or absence of individual taxonomic units within the SML. We conclude that physical stability of the SML below a system-specific wind speed threshold induces specific bacterial communities in the SML entailing strong implications for ecosystem functioning by wind-driven impacts on habitat properties, gas exchange and matter cycling processes.

Establishing the Secondary Metabolite Profile of the Marine Fungus: Tolypocladium geodes sp. MF458 and Subsequent Optimisation of Bioactive Secondary Metabolite Production.

As part of an international research project, the marine fungal strain collection of the Helmholtz Centre for Ocean Research (GEOMAR) research centre was analysed for secondary metabolite profiles associated with anticancer activity. Strain MF458 was identified as Tolypocladium geodes, by internal transcribed spacer region (ITS) sequence similarity and its natural product production profile. By using five different media in two conditions and two time points, we were able to identify eight natural products produced by MF458. As well as cyclosporin A (1), efrapeptin D (2), pyridoxatin (3), terricolin A (4), malettinins B and E (5 and 6), and tolypocladenols A1/A2 (8), we identified a new secondary metabolite which we termed tolypocladenol C (7). All compounds were analysed for their anticancer potential using a selection of the NCI60 cancer cell line panel, with malettinins B and E (5 and 6) being the most promising candidates. In order to obtain sufficient quantities of these compounds to start preclinical development, their production was transferred from a static flask culture to a stirred tank reactor, and fermentation medium development resulted in a nearly eight-fold increase in compound production. The strain MF458 is therefore a producer of a number of interesting and new secondary metabolites and their production levels can be readily improved to achieve higher yields.

Short-term molecular and physiological responses to heat stress in neritic copepods Acartia tonsa and Eurytemora affinis.

Invertebrates inhabiting shallow water habitats represent particularly appropriate organisms for studying the acclimation potential to environmental stress, since they naturally experience large fluctuations in key abiotic factors such as temperature and salinity. We quantified the biochemical- (mRNA transcripts of 78-kDa glucose-regulated protein (grp78), 70-kDa heat shock protein (hsp70), 90-kDa heat shock protein (hsp90), protein synthesis of HSP70) and organismal- (oxygen consumption rates) level responses to acute heat stress on two neritic copepods (Acartia tonsa and Eurytemora affinis) with special emphasis on the role of short-term acclimation. Transcripts of hsp increased with increasing acute temperature exposure and protein quantities (HSP70) were detectable for 30h. In A. tonsa, HSP70 synthesis was also associated with handling stress. In E. affinis, heat-dependent responses were detected in hsp90, grp78 (mRNA) and HSP70 (protein) expression. Acclimation to a warmer temperature significantly decreased the heat stress response in both species. In A. tonsa, short-term acclimation to heat was not detected at the organismal level via metabolic rate. This study reveals interspecific differences in both the gene expression of stress molecules (e.g. hsp90) as well as the stress factors needed to evoke a stress response (heat vs. handling). We demonstrate that cellular stress markers can be useful measures of short-term thermal acclimation in copepods, which may remain undetected by organismal-level measures.

Phenotypic detection of clonotypic B cells in multiple myeloma by specific immunoglobulin ligands reveals their rarity in multiple myeloma.

In multiple myeloma, circulating "clonotypic" B cells, that express the immunoglobulin rearrangement of the malignant plasma cell clone, can be indirectly detected by PCR. Their role as potential "feeder" cells for the malignant plasma cell pool remains controversial. Here we established for the first time an approach that allows direct tracking of such clonotypic cells by labeling with patient-specific immunoglobulin ligands in 15 patients with myeloma. Fifty percent of patients showed evidence of clonotypic B cells in blood or bone marrow by PCR. Epitope-mimicking peptides from random libraries were selected on each patient's individual immunoglobulin and used as ligands to trace cells expressing the idiotypic immunoglobulin on their surface. We established a flow cytometry and immunofluorescence protocol to track clonotypic B cells and validated it in two independent monoclonal B cell systems. Using this method, we found clonotypic B cells in only one out of 15 myeloma patients. In view of the assay's validated sensitivity level of 10(-3), this surprising data suggests that the abundance of such cells has been vastly overestimated in the past and that they apparently represent a very rare population in myeloma. Our novel tracing approach may open perspectives to isolate and analyze clonotypic B cells and determine their role in myeloma pathobiology.

Antigen-specificity of oligoclonal abnormal protein bands in multiple myeloma after allogeneic stem cell transplantation.

Myeloma patients may develop oligoclonal immunoglobulins, so-called abnormal protein bands (APB), after stem cell transplantation. APB do not correspond to the patient's paraprotein and confer a good prognosis. We set out to investigate whether such APB represent a humoral anti-myeloma immune response by screening immunoglobulins of 15 myeloma patients after allogeneic stem cell transplantation and a control group of healthy donors for reactivity with myeloma protein extracts. While the immunoglobulins of healthy donors did not react with myeloma protein extracts, patient-derived immunoglobulins showed variable levels of interaction, depending on the presence of APB on immunofixation. Most commonly, we detected interactions with heat-shock proteins, followed by neutral alpha-glucosidase, alpha-enolase and vimentin, as well as proliferating cell nuclear antigen and MAGEA4. More than 80% of targets were upregulated in myeloma. Heat-shock protein 60 (HSP60) was subsequently evaluated as an exemplary antigen. We found that HSP60 was aberrantly displayed on the surface of primary myeloma cells. Indeed, patient-derived APB-containing immunoglobulins recognized surface HSP60 suggesting that this antigen becomes accessible to the immune system after aberrant membrane exposition. We conclude that immunoglobulin fractions with APB recognize recurrent myeloma antigens and that this humoral response may contribute to the more favorable prognosis in patients with APB.