Marine viruses play a critical role not only in the global geochemical cycles but also in the biology and evolution of their hosts. Despite their importance, viral diversity remains underexplored mostly due to sampling and cultivation challenges. Direct sequencing approaches such as viromics has provided new insights into the marine viral world. As a complementary approach, we analysed 24 microbial metagenomes (>0.2 μm size range) obtained from six sites in the Mediterranean Sea that vary by depth, season and filter used to retrieve the fraction. Filter-size comparison showed a significant number of viral sequences that were retained on the larger-pore filters and were different from those found in the viral fraction from the same sample, indicating that some important viral information is missing using only assembly from viromes. Besides, we were able to describe 1,323 viral genomic fragments that were more than 10Kb in length, of which 36 represented complete viral genomes including some of them retrieved from a cross-assembly from different metagenomes. Host prediction based on sequence methods revealed new phage groups belonging to marine prokaryotes like SAR11, Cyanobacteria or SAR116. We also identified the first complete virophage from deep seawater and a new endemic clade of the recently discovered Marine group II Euryarchaeota virus. Furthermore, analysis of viral distribution using metagenomes and viromes indicated that most of the new phages were found exclusively in the Mediterranean Sea and some of them, mostly the ones recovered from deep metagenomes, do not recruit in any database probably indicating higher variability and endemicity in Mediterranean bathypelagic waters. Together these data provide the first detailed picture of genomic diversity, spatial and depth variations of viral communities within the Mediterranean Sea using metagenome assembly.

Background Lake Baikal is the largest body of liquid freshwater on Earth. Previous studies have described the microbial composition of this habitat, but the viral communities from this ecosystem have not been characterized in detail. Results Here, we describe the viral diversity of this habitat across depth and seasonal gradients. We discovered 19,475 bona fide viral sequences, which are derived from viruses predicted to infect abundant and ecologically important taxa that reside in Lake Baikal, such as Nitrospirota, Methylophilaceae, and Crenarchaeota. Diversity analysis revealed significant changes in viral community composition between epipelagic and bathypelagic zones. Analysis of the gene content of individual viral populations allowed us to describe one of the first bacteriophages that infect Nitrospirota, and their extensive repertoire of auxiliary metabolic genes that might enhance carbon fixation through the reductive TCA cycle. We also described bacteriophages of methylotrophic bacteria with the potential to enhance methanol oxidation and the S-adenosyl-L-methionine cycle. Conclusions These findings unraveled new ways by which viruses influence the carbon cycle in freshwater ecosystems, namely, by using auxiliary metabolic genes that act upon metabolisms of dark carbon fixation and methylotrophy. Therefore, our results shed light on the processes through which viruses can impact biogeochemical cycles of major ecological relevance.

Marine phages play a variety of critical roles in regulating the microbial composition of our oceans. Despite constituting the majority of genetic diversity within these environments, there are relatively few isolates with complete genome sequences or in-depth analyses of their host interaction mechanisms, such as characterization of their receptor binding proteins (RBPs). Here, we present the 92,760-bp genome of the Alteromonas-targeting phage V22. Genomic and morphological analyses identify V22 as a myovirus; however, due to a lack of sequence similarity to any other known myoviruses, we propose that V22 be classified as the type phage of a new Myoalterovirus genus within the Myoviridae family. V22 shows gene homology and synteny with two different subfamilies of phages infecting enterobacteria, specifically within the structural region of its genome. To improve our understanding of the V22 adsorption process, we identified putative RBPs (gp23, gp24, and gp26) and tested their ability to decorate the V22 propagation strain, Alteromonas mediterranea PT11, as recombinant green fluorescent protein (GFP)-tagged constructs. Only GFP-gp26 was capable of bacterial recognition and identified as the V22 RBP. Interestingly, production of functional GFP-gp26 required coexpression with the downstream protein gp27. GFP-gp26 could be expressed alone but was incapable of host recognition. By combining size-exclusion chromatography with fluorescence microscopy, we reveal how gp27 is not a component of the final RBP complex but instead is identified as a new type of phage-encoded intermolecular chaperone that is essential for maturation of the gp26 RBP. IMPORTANCE Host recognition by phage-encoded receptor binding proteins (RBPs) constitutes the first step in all phage infections and the most critical determinant of host specificity. By characterizing new types of RBPs and identifying their essential chaperones, we hope to expand the repertoire of known phage-host recognition machineries. Due to their genetic plasticity, studying RBPs and their associated chaperones can shed new light onto viral evolution affecting phage-host interactions, which is essential for fields such as phage therapy or biotechnology. In addition, since marine phages constitute one of the most important reservoirs of noncharacterized genetic diversity on the planet, their genomic and functional characterization may be of paramount importance for the discovery of novel genes with potential applications.

Graphical abstract

24Lake Baikal is the largest body of liquid freshwater on Earth. Previous studies have 25 described the microbial composition of this habitat but the viral communities from this ecosystem 26 have not been characterized in detail. Here we describe the viral diversity of this habitat across 27 depth and seasonal gradients. We discovered 19,475 bona fide viral sequences, which are derived 28 from viruses predicted to infect abundant and ecologically important taxa that reside in Lake Baikal, 29 2 such as Nitrospirota, Methylophilaceae and Crenarchaeota. Diversity analysis revealed significant 30 changes in viral community composition between epipelagic and bathypelagic zones. Analysis of 31 the gene content of individual viral populations allowed us to describe one of the first 32 bacteriophages that infect Nitrospirota, and their extensive repertoire of auxiliary metabolic genes 33 that might enhance carbon fixation through the reductive TCA cycle. We also described 34 bacteriophages of methylotrophic bacteria with the potential to enhance methanol oxidation and the 35 S-adenosyl-L-methionine cycle. These findings unraveled new ways by which viruses influence the 36 carbon cycle in freshwater ecosystems, namely by using auxiliary metabolic genes that act upon 37 metabolisms of dark carbon fixation and methylotrophy. Therefore, our results shed light on the 38 processes through which viruses can impact biogeochemical cycles of major ecological relevance. 39 40 reductive TCA cycle; Methylotrophy; S-adenosyl-L-methionine cycle 42 43 Phycodnaviridae (1,195), and Podoviridae (809). The presence of viruses of eukaryotes in our 132 dataset derives from the fact that samples were not pre-filtered to remove eukaryotic cells. 133Computational host prediction followed by manual curation allowed putative hosts at the taxonomic 134 level of domain to be assigned to 2,870 viral sequences. These predictions suggested that the 135 majority of these sequences belonged to viruses that infect Bacteria (2,135), but viruses that infect 136 Archaea (29), Eukaryotes (621) and even virophages (85) were also identified. Among those 137 assigned as viruses of bacteria (i.e. bacteriophages) the majority of sequences were predicted to 138 infect Actinobacteria (640), followed by Proteobacteria (375), Bacteroidota (241) and 139Cyanobacteria (226). Although less frequent, some sequences were predicted to be derived from 140