Environmental Microbiology (2018) 00(00), 00–00 doi:10.1111/1462-2920.14462 Novel Caudovirales associated with Marine Group I Thaumarchaeota assembled from metagenomes Mario López-Pérez ,1†* Jose M. Haro-Moreno,1† Church et al., 2003). This abundance and the discovery José R. de la Torre2 and Francisco Rodriguez-Valera1 that members of this lineage derive energy from the oxi- 1Evolutionary Genomics Group, dation of ammonia (Könneke et al., 2005) and are able to División de Microbiología, Universidad Miguel Hernández, fix inorganic forms of carbon (Berg et al., 2010) argue Apartado 18, San Juan, Alicante, 03550, Spain. that the marine Thaumarchaeota are important players in 2Department of Biology, San Francisco State University, global Carbon (C) and Nitrogen (N) biogeochemical San Francisco, CA, 94132, USA. cycles. Recent studies have shown that these marine archaea are responsible for the majority of the aerobic fi Summary nitri cation measured in marine environments and may be a significant source of the greenhouse gas nitrous Marine Group I (MGI) Thaumarchaeota are some of the oxide (Santoro et al., 2011). The cultivation of numerous most abundant microorganisms in the deep ocean and strains, as well as sequences from environmental meta- responsible for much of the ammonia oxidation occur- genomes and single-cell genomes have provided invalu- ring in this environment. In this work, we present able information on the ecology and evolution of this 35 sequences assembled from metagenomic samples diverse lineage (Luo et al., 2014; Swan et al., 2014; of the first uncultivated Caudovirales viruses associ- Santoro et al., 2015). However, despite these efforts, ated with Thaumarchaeota, which we designated remarkably little is known about their viruses. The vast marthavirus. Most of the sequences were obtained majority of archaeal viruses that have been isolated so from cellular metagenomes confirming that they repre- far came from either hyperthermophilic or hyperhalophilic sent an important tool to study environmental viral environments, where Crenarchaeota or Euryarchaeota communities due to cells retrieved while undergoing dominate (Snyder et al., 2015). Conversely, in the case viral lysis. Metagenomic recruitment showed that this viral population is formed by very divergent entities of the mesophilic archaea, only the advent of high- with high intrapopulation homogeneity. However, meta- throughput sequencing has provided novel information of transcriptomic analyses revealed the same differential the unknown viruses infecting archaea (Vik et al., 2017; expression profile with the capsid as major transcript, Roux et al., 2016), expanding viral diversity far beyond indicative of viruses during the lytic cycle. The cobala- that established by traditional methods for virus isolation. mine biosynthesis gene cobS, an auxiliary metabolic The recently discovered Marine Group II Euryarchaeota gene, was also highly expressed during the infection. viruses (magrovirus) group from assembled sequences These analyses expand our understanding of the (Philosof et al., 2017), which infects the ubiquitous and global diversity of archaeal viruses. abundant but yet uncultured Marine Group II Euryarch- aeota, is an example of the benefits of metagenomics. Introduction However, to date no marine thaumarchaeal virus has yet been isolated probably because their host are also diffi- Marine Thaumarchaeota, initially discovered 26 years cult to obtain in pure culture. Only two putative viral ago by 16S rRNA gene surveys (Fuhrman and McCal- sequences have been retrieved by single-cell genomics lum, 1992; Delong, 1992), are some of the most abun- (Labonté et al., 2015) and fosmid libraries (Chow et al., dant microorganisms in the deep ocean, accounting for 2015). Additionally, a putative provirus has been found up to 40% of the bacterioplankton below the euphotic within the genome of Ca. Nitrosomarinus catalina zone (Karner et al., 2001; Fuhrman and Ouverney, 1998; SPOT01 (Ahlgren et al., 2017). Previous studies showed that viruses infecting Thaumarchaeota from the deep Received 19 September, 2018; revised 23 October, 2018; accepted ocean were more active than bacterial viruses, contribut- 24 October, 2018. *For correspondence. E-mail [email protected]; Tel. +34-965919313; Fax +34-965 919457. †These authors contributed ing to their cell lysis and, hence, modifying the biogeo- equally to this work. chemical cycles of N and C (Danovaro et al., 2016). © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd. 2 M. López-Pérez, J.M. Haro-Moreno, J.R. de la Torre and F. Rodriguez-Valera In this work, we present 35 sequences assembled from expeditions (Duarte, 2015) and datasets publicly avail- metagenomic samples of the first uncultivated viruses able at the Joint Genome Institute (JGI) database (https:// associated with marine Thaumarchaeota. Sequences of img.jgi.doe.gov/). In the end, we identified 35 putative this newly identified viral population are locally distributed viral contigs (Supporting Information Table S1) manually with low intra-population diversity. Metatranscriptomic curated to check for similarity to these reference proteins analyses showed that they were retrieved while undergo- and thaumarchaeal genes (see Material and Methods). ing viral lysis and apart from the capsid, the essential Most contigs with similarity to these proteins (#18) were structural component, expression of the cobalamine bio- found in our own dataset (Med-OCT2015-75m and Med- synthesis gene cobS, an auxiliary metabolic gene, was OCT2015-90m), 17 in the cellular and one in the viral also high during the infection. Our analyses provide fraction (MedVir-OCT2015-60m). Interestingly, another important insights into the genomic diversity of this new large batch (9 genomes) was found in viromes from the marine viral population, which remain uncultivated, Chesapeake Bay, an estuary where strong ammonia gra- expanding our understanding of the global diversity of dients are also found (Maresca et al., 2018). All had a archaeal viruses. GC content varying from 30% to 37%, as expected from the low-GC content of marine Thaumarchaeota genomes (Ahlgren et al., 2017; Supporting Information Table S1). Results and discussion Based on the presence of terminal inverted repeats >30 Recently, we characterized variations in the marine nucleotides only two viral genomes were complete. A microbiome at different depths within the photic zone dur- total of 1289 open reading frames were identified in all ing a period of strong thermal stratification of the water the sequences. However, only 14% showed significant column (Haro-Moreno et al., 2018). Results showed that homology to sequences present in the pVOGs marine Thaumarchaeota were only found below the deep (Prokaryotic Virus Orthologous Groups) database chlorophyll maximum (accounted for up to 10% of the (Grazziotin et al., 2017), as typical for novel viruses. community at 90 m), coinciding with the increase of avail- Clustering of the sequences based on similarity resulted able ammonia that is practically non-existent at shallower in 684 protein clusters, 11 of which formed the viral ‘soft’ depths. Analyses of the assembled contigs from these core (they were present in at least half of the sequences) samples showed the presence of a 69 kb contig that had (Supporting Information Table S2). Five of the ‘soft’ core hits to a few Thaumarchaeota genomes (the majority of protein clusters contained proteins involved in DNA these hits were to the genus Ca. Nitrosopumilus) but also metabolism (terminase, RadA, ATPase, PD-D/EXK to viral-related genes, including predicted major capsid nuclease and Ribonuclease H) and one sequence was proteins (MCP), portal proteins, tail tape measure pro- an auxiliary metabolic gene (AMG), cobS, that encodes a teins and the large subunit of viral terminases. Both the protein that catalyses the final step in cobalamin (vitamin terminase and the MCP proteins gave hits with low iden- B12) biosynthesis in prokaryotes, which has previously tity (32–35%) to a complete, unclassified archaeal virus only been found in cyanophages (Sullivan et al., 2005). (KY229235) recovered from a metagenomic assembly of Unfortunately, the remaining five ‘soft’ core clusters were a sample ~550 m below the seafloor (Nigro et al., 2017). hypothetical proteins and no function could be inferred. Like in the case of KY229235, our contig had identical Furthermore, no tRNA-encoding sequences or hallmarks repeated sequences (>30 nucleotides) at the 50 and 30 of temperate phage, such as integrase or excisionase terminal regions suggesting a complete viral genome. It genes, were detected in any of the recovered genomes. has been demonstrated that cellular metagenomes In addition to the terminase, we found other clusters indi- (>0.22 μm size fraction) are a source of bacterial and cating the Caudovirales affiliation of these viruses such archaeal viruses that are undergoing the lytic cycle and as prohead or portal proteins. This is to our knowledge actively replicating their DNA (López-Pérez et al., 2017). the first group of head-tail viruses described for the Cre- narchaeal superphylum, although they are relatively com- mon in Euryarchaea (Rachel et al., 2002; Pietilä New Thaumarchaeota viruses recovered from et al., 2014). metagenomic samples In order to expand the repertoire of putative Thaumarch- Phylogeny and host assignment aeota viruses we used the MCP, terminase and portal proteins of this new contig and KY229235 sequences as We next sought to establish