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Home , Ampullaviridae, Marine viruses, Nitrosopumilus, Salterprovirus

Spindle-shaped infect marine - oxidizing thaumarchaea

Jong-Geol Kima, So-Jeong Kimb, Virginija Cvirkaite-Krupovicc, Woon-Jong Yua, Joo-Han Gwaka, Mario López-Pérezd, Francisco Rodriguez-Valerad, Mart Krupovicc, Jang-Cheon Choe, and Sung-Keun Rheea,1

aDepartment of , Chungbuk National University, Heungduk-gu, 361-763 Cheongju, South Korea; bGeologic Environment Research Division, Korea Institute of Geoscience and Mineral Resources, 34132 Daejeon, Republic of Korea; cDepartment of Microbiology, Institut Pasteur, 75015 Paris, France; dEvolutionary Genomics Group, Universidad Miguel Hernandez, San Juan, 03540 Alicante, Spain; and eDepartment of Biological Sciences, Inha University, 22212 Incheon, Republic of Korea

Edited by Edward F. DeLong, University of Hawaii at Manoa, Honolulu, HI, and approved June 21, 2019 (received for review April 3, 2019) Ammonia-oxidizing (AOA) from the viruses of the order have been previously identified in are ubiquitous in marine and play a prominent role in the of the soil thaumarchaeon Nitrososphaera viennensis carbon and nitrogen cycling. Previous studies have suggested that, (9) and the extremely thermophilic thaumarchaeon Candidatus like all microbes, thaumarchaea are infected by viruses and that viral Nitrosocaldus cavascurensis (10). Furthermore, several meta- predation has a profound impact on thaumarchaeal functioning and genomic and single- genomic studies have resulted in the as- mortality, thereby regulating global biogeochemical cycles. However, sembly of putative AOA genomes, all related to members of not a single virus capable of infecting thaumarchaea has been the order Caudovirales (11–13). Notably, some of these assembled reported thus far. Here we describe the isolation and characterization virus genomes were found to carry putative genes encoding the of three spindle-shaped viruses (NSVs) that infect ammonia monooxygenase subunit C (amoC), a key component of AOA and are distinct from other known . Although ammonia monooxygenase (AMO) (13, 14), suggesting an active role NSVs have a narrow range, they efficiently infect autochtho- of viruses in nitrogen cycling in the oceans. Nevertheless, not a single nous Nitrosopumilus strains and display high rates of adsorption thaumarchaeal virus–host system has been isolated or cultivated thus to their host cells. The NSVs have linear double-stranded DNA ge- far, precluding functional studies on the virus–host interactions and ∼ nomes of 28 kb that do not display appreciable sequence simi- the effect of viruses on the metabolic activity of thaumarchaea. MICROBIOLOGY larity to genomes of other known archaeal or bacterial viruses and Archaea are associated with a remarkably diverse virosphere, could be considered as representatives of a new virus family, the which is characterized by unique morphotypes not observed “Thaspiviridae.” Upon infection, NSV replication leads to inhibition among viruses infecting and Eukarya. These include of AOA growth, accompanied by severe reduction in the rate of am- virions with spindle-shaped, bottle-shaped, droplet-shaped, coil- monia oxidation and nitrite reduction. Nevertheless, unlike in the case shaped, and other morphologies (15–18). Among these archaea- of lytic , NSV propagation is not associated with de- specific morphotypes, spindle-shaped viruses are among the tectable degradation of the host chromosome or a decrease in cell most widely distributed (19) and were found not only in extreme counts. The broad distribution of NSVs in AOA-dominated marine geothermal and hypersaline habitats, but also in marine environments, environments suggests that NSV predation might regulate the diver- sity and dynamics of AOA communities. Collectively, our results Significance shed light on the diversity, , and potential impact of the virosphere associated with ecologically important mesophilic archaea. Ammonia-oxidizing archaea (AOA) are major players in global spindle-shaped virus | ammonia-oxidizing archaea | viral predation | nitrogen cycling. The physicochemical and metabolic factors af- chronic infection fecting the composition of AOA communities and their efficiency of resource utilization have been studied extensively. However, viral predation on AOA remains unexplored due to lack of iso- embers of the phylum Thaumarchaeota are widespread lated virus–host systems. Here we report on the isolation and Mand abundant in marine ecosystems and play key roles in characterization of three Nitrosopumilus spindle-shaped viruses nitrogen cycles by mediating ammonia oxidation (1, 2). Ammonia (NSVs) that infect AOA hosts. NSVs represent a potentially im- oxidation is implicated in controlling the availability of nitrogen portant group of marine viruses with a chronic infection cycle, , production of N2O (3, 4), and is associated with carbon providing important insights into the diversity and evolution of fixation in the deep ocean. Thus, information on key factors af- the archaeal virosphere. The wide spread of NSVs in AOA- fecting abundance and composition of the communities of containing marine environments suggests that NSV predation ammonia-oxidizing archaea (AOA) is crucial for understanding might regulate the diversity and dynamics of AOA communities, the biogeochemical processes of nitrogen cycling in the oceans. thereby affecting the carbon and nitrogen cycling. The relative contribution of resource competition (bottom-up) and predation (top-down control) are the key drivers of bio- Author contributions: J.-G.K. and S.-K.R. designed research; J.-G.K., V.C.-K., W.-J.Y., and geochemical cycles, affecting microbial activity and community J.-H.G. performed research; S.-J.K. and M.L.-P. contributed new reagents/analytic tools; S.-J.K., V.C.-K., M.L.-P., F.R.-V., M.K., and J.-C.C. analyzed data; and J.-G.K., F.R.-V., M.K., structures. To understand the abundance and composition of the and S.-K.R. wrote the paper. AOA communities, the effects of physicochemical factors and The authors declare no conflict of interest. metabolic traits of AOA ecotypes on the efficiency of resource This article is a PNAS Direct Submission. utilization have been thoroughly assessed (2, 5). Published under the PNAS license. Predation pressure can also influence AOA communities but Data deposition: DNA sequencing data have been deposited in the GenBank database has been rarely studied. Flagellate grazing was proposed to affect with the identifiers MK570053 to MK570059. Accession number of the Nitrosopumilus the distribution and abundance of AOA in planktonic microbial strain SW is CP035425. assemblages (6, 7). Danovaro et al. (8) suggested that viral in- 1To whom correspondence may be addressed. Email: [email protected]. fection represents a key mechanism controlling the turnover of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. archaea, especially AOA, in surface deep-sea sediments. Putative 1073/pnas.1905682116/-/DCSupplemental. thaumarchaeal proviruses related to tailed bacterial and archaeal Published online July 16, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1905682116 PNAS | July 30, 2019 | vol. 116 | no. 31 | 15645–15650 Downloaded by guest on September 27, 2021 Table 1. General features of NSVs and scaffolds related to NSV Virus/putative viral Adsorption Latent Attached Genome Number AOA-like Accession Isolation site scaffolds* rate, 50% (min) period (h) fraction (%) size (kb) of ORFs G+C mol% genes number

Bulcheon NSV1 5 6 69 27.5 48 29.8 5 MK570053 (36°57′N, 126°20′E) NSV2 10 6–8 80 28.9 51 29.8 4 MK570055 Scaffold83 ———14.6 30 27.1 1 MK570056 Scaffold98 ———13.4 20 31.3 1 MK570057 Scaffold261 ———7.1 18 29.7 0 MK570058 Scaffold342 ———6.0 13 27.2 2 MK570059 Daecheon NSV3 <5 6 70 27.5 48 29.8 5 MK570054 (36°58′N, 126°20′E)

—, not applicable. *Scaffold is obtained from early phase enrichment culture for NSV2.

including surfaces (20), particulate matter-rich bays (21), and the and temperatures up to 55 °C (SI Appendix, Fig. S4). These re- oceanic basement (22), although their hosts and genome sequences sults showed that NSV virions were well-adapted to both survive were not determined. environmental fluctuations and interact with the unique archaeal In this study, we isolated and characterized spindle-shaped cell surface, which consists of a cytoplasmic membrane and viruses infecting AOA from coastal seawater and revealed proteinaceous S-layer (32–35). Notably, mesophilic AOA are properties of their cycles. We show that virus infection has a believed to have evolved from a (hyper)thermophilic ancestor dramatic effect on ammonia oxidation and is likely to affect the (36, 37). Thus, the observed resilience of the NSV particles could population structure and functioning of the AOA community. Our have been inherited from an ancestral extremophilic virus. results shed light on the diversity, evolution, and potential impact of the virosphere associated with ecologically important archaea. Host Specificity. Based on the comparison of average nucleotide identities (ANIs), strain SW belongs to the Nitrosopumilus, Results and Discussion but represents a species that is most closely related to Nitro- sopumilus maritimus SI Appendix Isolation, Morphology, and Stability. Three virus strains, designated SCM1 ( , Fig. S5). Thus, host Nitrosopumilus spindle-shaped viruses 1, 2, and 3 (NSV1, NSV2, specificities of the three NSVs were tested using strains of Nitrosopumilus > and NSV3, respectively), were isolated from suspended partic- species closely related to the strain SW ( 98% SI Appendix ulate matter (SPM)-rich seawater samples taken from the west- sequence similarity of the 16S rRNA gene; , Fig. S1) ern coast of the Korean Peninsula using as a host the axenic SCM1 (33), DDS1 (23), HCA1 (33), and BC. In the presence of AOA strain SW (SI Appendix, Fig. S1), which was isolated from NSVs, neither virus production nor inhibition of ammonia oxi- surface water (20 m deep) at the Yellow Sea, Korea (23) (see dation by these AOA strains was observed, indicating that the below for further information). Since AOA could not form lawns host range of NSVs might be rather narrow. The sensitivity of on agar plates, the dilution-to-extinction method was used to closely related strains to viral infection could be potentially af- isolate the viruses from enrichment cultures. General features of fected by the presence of host defense mechanisms or a lack of specific receptors on the cell surface. To date, there is no report NSVs are summarized in Table 1. The sizes of the spindle- – shaped NSV virions were similar (Fig. 1 and SI Appendix, Fig. of a CRISPR-Cas dependent viral defense system in the thau- marchaeal group I.1a (38). Similarly, homologs of the Dnd de- S2), measuring 64 ± 3 nm in diameter and 112 ± 6 nm in length, fense system could not be identified in the available genomes of with a short tail at one pole (Fig. 1A). The morphological fea- AOA strains (i.e., strains SCM1, DDS1, and SW) (39) used in this tures of NSVs were very similar to those of viruses in the family study. These findings suggest that resistance might instead be due and in the genus , which infect to the absence or modification of cell surface receptors. Notably, hyperthermophilic and hyperhalophilic archaea, respectively (19, comparison of the closely related Nitrosopumilus maritimus SCM1 24, 25). A large fraction of produced virions remained at- Nitrosopumilus B and sp. SW genomes revealed that genomic island 2 tached to the cell surface (Fig. 1 ). Elongation of virions into predictedtobeinvolvedincellsurface modification had different arrowhead-shaped particles with long tails was observed 6 h post C gene content in the two strains, which might explain the different infection (Fig. 1 ), suggesting that flexibility of virions might be susceptibility to NSVs (SI Appendix,Fig.S6and Dataset S1). important to the infection process—as has been observed for spindle-shaped virions of the fusellovirus SSV1 (26) and the bicaudavirus ATV (27). The adsorption of NSVs to AOA cells was rapid, with ∼50% of virions bound to cell surfaces within 10 AB C min (SI Appendix, Fig. S3). Certain hyperthermophilic archaeal viruses exhibit comparably rapid adsorption kinetics (28); con- versely, halophilic archaeal viruses—including those with spindle- shaped virions—generally exhibit slow adsorption kinetics (29). Spindle-shaped viruses are frequently observed in extreme environments, and it is suggested that this morphotype has been selected for its robustness under a wide range of extreme envi- ronmental conditions (24, 30, 31). To study how the isolated Fig. 1. Transmission electron microscopy images of negatively stained NSV1 NSV virions respond to physicochemical fluctuations in the en- virions. (A) NSV1 virions. (Scale bar, 50 nm.) (B) NSV1 particles attached to vironment, their stabilities were tested under varying regimes of the surface of an SW cell. (Scale bar, 200 nm.) (C) Elongated NSV1 particles pH, salinity, and temperature. NSV virions remained stable and attached to the surface of an SW cell after 12 h of infection. The arrows infectious between pH 3 and 9, salinities between 0.1 and 20%, indicate elongated NSV1 particles. (Scale bar, 200 nm.)

15646 | www.pnas.org/cgi/doi/10.1073/pnas.1905682116 Kim et al. Downloaded by guest on September 27, 2021 Genome Analysis. The genomes of NSV1, NSV2, and NSV3 of the infection cycle cannot be ruled out. Similarly to many consist of linear dsDNA molecules of ∼27, ∼29, and ∼27 kb, other archaeal viruses (40), NSV1 encodes two predicted gly- respectively, containing 176-bp terminal inverted repeats (Table 1). cosyltransferases (ORF20 and ORF39), with the closest homo- The numbers of ORFs in the genomes of NSV1, NSV2, and logs present in thaumarchaeal genomes, and one predicted DNA NSV3 were predicted to be 48, 51, and 48, respectively. The methyltransferase (ORF33)—apparently recruited from bacteria NSV1 and NSV3 genomes were highly similar (ANI = 99.8%; by . ORFs 6, 7, 29, 31, 32, and 37 encode Fig. 2), whereas the NSV2 genome was slightly more divergent short with predicted zinc-binding domains; ORFs 18, 26, (ANI = 95%; Fig. 2). Divergent partial NSV genomes were also and 44 encode putative DNA-binding proteins with winged obtained as scaffolds 83, −98, −261, and −342 from meta- helix-turn-helix, looped-hinge helix, and ribbon-helix-helix do- genomes of the initial NSV2 enrichment culture, indicating a mains, respectively (Dataset S2). To gain further understanding on greater diversity of NSVs (Fig. 2). Despite being closely related the proteins encoded by NSVs, purified NSV1 virions were to each other, NSVs did not display appreciable sequence simi- subjected to proteomic characterization by liquid chromatography larity to other known archaeal or bacterial viruses in BLASTP coupled to tandem mass spectrometry (LC-MS/MS). Ten NSV1 searches (E value cutoff: 0.001; Dataset S2). Indeed, none of the proteins were detected by proteomic analysis (Dataset S3). NSV ORFs gave BLAST best-hits to known viral proteins, and only 9 out of 48 (18.7%) NSV1 ORFs yielded significant matches Evolutionary Relationships to Other Archaeal Viruses. Despite the in the nonredundant sequence databases to known cellular lack of direct sequence similarity, general features of the genome proteins—a common trend in archaeal viruses (15). Five of the organization and proteome of NSV1 are reminiscent of those of nine hits were to proteins encoded by various marine thau- other archaeal viruses. In particular, the pPolB of NSVs is shared marchaea, and four were to bacterial proteins (Dataset S2). The with several groups of archaeal viruses and nonviral mobile ge- thaumarchaea-like ORFs encode putative genome replication netic elements, which, like NSV, have linear genomes with terminal proteins (ORF3 and ORF43), glycosyltransferases (ORF20 and inverted repeats. These include the haloarchaeal spindle-shaped ORF39), and a DNA-binding (ORF44) (see below). (genus Salterprovirus) and pleomorphic (family ,ge- Accordingly, NSVs might be considered as representatives of a nus Gammapleolipovirus) viruses His1 (41) and His2 (30), re- new archaeal virus family with the proposed name “Thaspiviridae” spectively; hyperthermophilic bottle-shaped (family ) (for Thaumarchaeal spindle-shaped viruses). (25) and ellipsoid (family Ovaliviridae) (42) viruses; and casposons, More sensitive sequence analysis based on profile hidden which integrate into the genomes of diverse thaumarchaea (43, 44). Markov model (HMM) comparisons allowed for functional an- To understand the relationship between NSVs and other pPolB- MICROBIOLOGY notation of 15 putative NSV1 genes (Dataset S2). ORF3 and encoding archaeal and bacterial mobile genetic elements, a maxi- ORF43, respectively, encode predicted protein-primed family B mum likelihood phylogenetic analysis of their respective pPolB DNA (pPolB) and a DNA sliding clamp known as sequences was performed. When midpoint rooted, a well-supported proliferating cell nuclear antigen (PCNA) that are likely to be phylogeny splits between bacterial and archaeal sequences, with the involved in NSV genome replication. ORF15 encodes a pre- only exception being the ovalivirus SEV1, which groups with bac- dicted Cdc6-like AAA+ ATPase that may also be involved in terial rather than archaeal homologs (Fig. 3A). The pPolB se- genome replication. However, given the broad functional di- quences from NSVs form a sister group to the clade that includes versity of AAA+ ATPases, involvement of ORF15 in other steps halophilic viruses His1 and His2, as well as sequences from marine

Fig. 2. Comparative genomics of NSVs. Genomic maps of NSVs and related scaffolds. Shared ORFs are connected by color-coded shaded areas based on sequence identity. Genomes of NSVs are flanked by terminal inverted repeats. %GC represents mol% G+C content of DNA.

Kim et al. PNAS | July 30, 2019 | vol. 116 | no. 31 | 15647 Downloaded by guest on September 27, 2021 sediment metagenomes. At the base of this clade are casposons groups. Notably, despite the lack of detectable sequence similarity, from the marine Thaumarchaeota.Interestingly,G+Cmol%values the two viruses possess a similar gene repertoire (Fig. 3B), including of His1 (39%) and His2 (40%) are different from that of their host, many genes encoding zinc-binding proteins, AAA+ ATPase, gly- hispanica (63%), but close to those of AOA (∼34%). cosyltransferases, and a putative terminal protein which is found Collectively, these results suggest horizontal exchange of the pPolB by LC-MS/MS in the virions of both NSV1 (Dataset S3) and His1 genes between casposons, NSVs, and haloarchaeal viruses. Fur- (45). Furthermore, all known spindle-shaped viruses—including — – thermore, phylogenetic analysis suggests that pPolB genes of His1 encode relatively short (70 140 aa) major proteins α thaumarchaeal mobile elements are ancestral to those of His1-like containing two highly hydrophobic -helical regions predicted to viruses of halophilic archaea. However, many more viral genomes form transmembrane domains (19), but with little sequence sim- ilarity to each other. Among NSV1 proteins, only one, encoded by of both His1-like and NSV-like viruses are needed to substantiate SI Appendix this hypothesis. ORF12, fits these characteristics ( ,Fig.S7) and was NSVs and the salterprovirus His1 are the only known spindle- detected in the virions by LC-MS/MS (Dataset S3). Based on these shaped viruses with linear dsDNA genomes carrying pPolB genes, shared properties, NSVs appear to be distantly related to His1 and, more generally, to other spindle-shaped archaeal viruses. suggesting a specific evolutionary connection between the two virus All previously characterized archaeal viruses with unique vi- rion morphologies not observed among viruses of bacteria or infect extremophilic hosts (15, 25), suggesting that A these archaea-specific morphotypes have evolved as an adap- 99 63 (phi29-like phages) tation to extreme environments. The fact that genomes of 100 thaumarchaeal viruses previously discovered by 96 Tectiviridae (monoderm hosts) 99 are all related to those of tailed bacteriophages (11–13) is con- Tectiviridae (diderm hosts) sistent with this possibility. However, the identification of spindle- 100 "Autolykiviridae" shaped viruses infecting mesophilic marine thaumarchaea strongly 100 ATY46514.1_Sulfolobus ellipsoid virus 1 suggests that the spread of unique archaeal morphotypes extends to Ovaliviridae mesophilic archaea and possibly encompasses other archaeal line- 94 MGYP000338936204 ages. Furthermore, these results suggest that spindle-shaped viruses 100 MGYP000401327420 100 have a deep evolutionary history within the domain Archaea, which 100 candidate archaeal division MSBL1 likely dates back to the last archaeal common ancestor. metagenomic sequences 99 100 Ampullaviridae Viral Impact on Host Metabolism. The effects of NSVs on the physiology and metabolism of their AOA host were examined 100 100 Thaumarchaeal casposons (marine) next. During the first 2 d post infection (dpi), viral DNA repli- 99 NSV1_gp3 cation occurred concurrently with AOA growth and was ac- 99 SI 88 NSV2_gp4 Nitrosopumilus companied by a normal rate of ammonia oxidation (Fig. 4 and Appendix, Fig. S8). However, host cell growth ceased 2 dpi, while 100 NSV3_gp3 spindle--shaped viruses 99 100 ammonia oxidation continued until 4 dpi at a rate similar to that scaffold98_gp4 in the uninfected AOA (Fig. 4 and SI Appendix, Fig. S8). Pre- scaffold83_gp11 sumably, during this period, the energy obtained from ammonia 97 YP_529524.1_His1 Salterprovirus oxidation was directed to virus replication, consistent with the Gamma- 92 100 YP_529644.1_His2 observed increase in the viral titer until 6 dpi. However, after 5 pleolipovirus 100 MGYP000367844654 dpi, the rate of ammonia oxidation and nitrite production de- creased dramatically, indicating that NSVs had a severe effect on MGYP000577203607 Marine metabolic activity in their AOA hosts. Notably, virus production 99 MGYP000041105224 sediment metagenome was not associated with detectable degradation of the host 100 MGYP000220981334 chromosome determined by quantification of archaeal 16S rRNA gene (Fig. 4B) or a decrease in cell counts measured using epifluorescence microscopy (SI Appendix, Fig. S9). Consistent B with this observation, cells with damaged cell envelopes, as ob- served for some lytic archaeal viruses (46), were not detectable by transmission electron microscopy (TEM). Instead, virions were observed in abundance on the cell surface without obvious asso- ciated perturbations (Fig. 1B), suggesting that and release did not lead to cell . It has been previously shown that Fig. 3. Phylogeny of pPolB and comparative genome maps of NSV1 and spindle-shaped viruses of hyperthermophilic and halophilic ar- His1. (A) Maximum likelihood phylogenetic analysis of pPolB sequences from chaea are also released from their hosts without causing cell lysis bacterial and archaeal viruses. In this tree, archaeal viruses comprise the Sulfolobus following taxa: Ampullaviridae, Pleolipoviridae, Ovaliviridae,andSalterprovirus. (26, 47). In the case of spindle-shaped virus SSV1, vi- Sequences originating from marine environments and hyperhalophilic rions are assembled during the budding of the viral nucleoprotein archaeal viruses are highlighted with light blue and green backgrounds, through the cell membrane, which remains intact, in a process respectively. Sequences from metagenomic datasets are indicated with highly similar to the egress of enveloped eukaryotic viruses (26). gray font. (B) Comparison of the NSV1 and His1 genome maps. Function- We hypothesize that NSV virions are assembled and released ally equivalent genes are indicated with matching colors. Genes encoding from the cell by a similar mechanism without lysing the host cells. proteins detected in the purified virus particles are shown in cyan. Genes As suggested by the TEM analysis (Fig. 1B) and the adsorp- encoding small proteins containing Zn-binding domains are shown in tion assays (SI Appendix, Fig. S3), high proportions (>60%) of green. Abbreviations: pPolB, protein-primed family B DNA polymerase; MCP, major capsid protein (putative); wHTH, winged helix-turn-helix; GTase, gly- the produced NSVs remained cell-associated, presumably both cosyltransferase; MTase, DNA methyltransferase; PCNA, proliferating cell as adsorbed virions on the host cell surface and as intracellular nuclear antigen; RHH, ribbon-helix-helix. The question mark next to the replicated genome copies (Fig. 4C and SI Appendix, Fig. S8 C putative MCP denotes the uncertainty of this prediction. and D). In resource-poor oceans, a nonlytic mode of replication

15648 | www.pnas.org/cgi/doi/10.1073/pnas.1905682116 Kim et al. Downloaded by guest on September 27, 2021 and high adsorption rate might represent an optimal strategy for survival of viruses infecting chemolithoautotrophic AOA hosts. The total number of NSV particles produced under laboratory conditions was ∼298 ± 18 virions per AOA cell at 6 dpi and ∼ 3 A 1.0 10 virus particles per micromole of NH3 oxidized, with slight strain-specific variations (Fig. 4 and SI Appendix,Fig.S8). The )Mm(etirtindnaainommA nonlytic replication strategy of NSVs and other spindle-shaped ar- 0.8 chaeal viruses (26, 47), which allows for continuous virion production and release, is radically different from the lytic life cycle of tailed viruses of the order Caudovirales (48) but, in certain aspects, re- 0.6 Ammonia (NSV1-infected) sembles the nonlytic production of filamentous bacteriophages of the Ammonia (Non-infected control) Inoviridae family (49). Consequently, conventional ecological Nitrite (NSV1-infected) models which are tailored to the mode of bacterial predation by Nitrite (Non-infected control) 0.4 lytic head–tail phages (50, 51) might benefit from revision ac- counting for alternative virus life cycles—exemplified here by NSVs and the Nitrosopumilus sp. SW. 0.2 Environmental Distribution. Distribution of NSVs in marine envi- ronments was analyzed using quantitative real-time PCR of the 0.0 NSV-specific pPolB gene. NSVs were detected in abundance in various marine sediments (104–106 NSV genome copies per gram of B 4 6 10 sediment), coastal seawaters (10 –10 NSV genome copies per liter ) 10 4 -1 ∼ lm(rebmunypocenegBloP of seawater), and coral-rich seawater ( 10 NSV genome copies per liter of seawater) (SI Appendix,TableS1). By contrast, NSVs were below the detection limit in a liter of typical oligotrophic seawater,

dnaenegANRrS61 109 consistent with a previous study showing that spindle-shaped virus

particles corresponded to <1% of virions in tested seawater samples MICROBIOLOGY (52). A comparison of the ratio of AOA counts to NSV counts AO A (Pearson’s correlation coefficient 0.557; P value 0.015) indicated 8 10 Total NSV1 that NSV levels might be positively correlated with the abundance of AOA present. However, the correlation of counts of strain SW- specific gene slp2 encoding an S-layer protein to those of NSV counts was weak, suggesting that viral host specificity might be as- 7 10 sociated to the different versions of this protein (53). p

lariV Metagenomic recruitment analysis showed that NSVs were not detected in any of the available metaviromes from marine envi- 106 ronments including those from sediments. A high adsorption rate of NSV particles to host cells and particulate matter (Figs. 1B and 4C) C 100 might have caused low recovery of NSVs from the viral fraction of the sediments during virome preparations, which could explain the lack of reads matching NSVs in the public metaviromes. Indeed, this )%( 80 possibility was confirmed experimentally: only ∼1% of NSV particles

surivdedn present in marine sediments could be extracted into the viral frac- tion using the approach commonly used for virome preparation (SI 60 Appendix,Fig.S10). The adsorption of NSV to particulate matter is also evidenced by frequent observation of NSV-like morphotypes in

e SPM-rich bays (21) and surface microlayers of (20). Thus, psus 40 NSV-like genomes could be underrepresented in the viral fractions extracted by conventional means from marine environments.

ylee In addition to resource competition (bottom-up control), predation 20 (top-down control) can act as a key driver of biogeochemical cycles by rF affecting microbial activity and community structures. Thus far, a virus capable of infecting thaumarchaea had not been isolated, which 0 had limited the fundamental knowledge of the impact of viral in- 0 2 4 6810 12 14 fection on the functioning and mortality of AOA. In this study, spindle-shaped viruses were found to infect a marine ammonia- Time (days) oxidizing thaumarchaeon. The genome architecture and life cycle of Fig. 4. Properties of the NSV1 infection cycle. Strain SW cells were in- the examined NSVs indicate that they are distantly related to spindle- fected with NSV1. Error bars represent SDs for three biological repli- shaped viruses that infect hyperthermophilic and hyperhalophilic ar- cates. (A) Comparison of ammonia oxidation by NSV1-infected and chaea but represent a distinct new viral family. Characterization of noninfected control cells. (B) Virus production by strain SW cells infected the infection strategy employed by NSVs suggests they might be with NSV1. AOA growth and virus production were measured by qPCR adapted for efficient infection of chemolithoautotrophic AOA hosts quantification of 16S rRNA and pPolB genes, respectively. (C)Fractionof nonadsorbed NSV1 virions, estimated by qPCR of viral pPolB gene. Viral and survival in resource-poor oceans. This study provides evidence genomic DNA of nonadsorbed virions was prepared from the culture that viral predation severely affects the metabolism of infected AOA supernatant. cells, with a potential impact on global carbon and nitrogen cycling.

Kim et al. PNAS | July 30, 2019 | vol. 116 | no. 31 | 15649 Downloaded by guest on September 27, 2021 Materials and Methods ACKNOWLEDGMENTS. This research was supported by National Research Foundation of Korea (NRF) grants (Mid-Career Researcher Program [NRF- Isolation of AOA viruses, analysis of infection cycle of NSVs, and sequencing 2018R1A2B6008861], Basic Research Laboratory Program [NRF-2015R1A4A1041869], and annotation of viral genomes are described in SI Appendix, SI Materials and C1 Gas Refinery Program [NRF-2015M3D3A1A01064881]) funded by the and Methods. Details of DNA extraction from seawater and marine sedi- Ministry of Science, Information and Communication Technology, and Future ment, quantification of AOA and NSV from marine environments, and Planning. M.K. was supported by a grant from l’Agence Nationale de la Recherche metagenomic read recruitments are provided in SI Appendix, SI Materials (#ANR-17-CE15-0005-01). The authors would like to thank Thibault Chaze and and Methods. Adsorption, host range, and stability of NSVs were tested as Mariette Matondo (Pasteur Proteomics Platform) for help with the proteomics analyses. M.L.-P. was supported by a postdoctoral fellowship (Juan de la Cierva) described in SI Appendix, SI Materials and Methods. Phylogenetic analysis from the Spanish Ministerio de Economía y Competitividad (IJCI-2017-34002). of pPolB, comparative genomic analysis of AOA strains and NSVs, and F.R.-V. was supported by grant CGL2016-76273-P (Agencia Estatal de Investiga- proteome analysis of NSV1 are described in SI Appendix, SI Materials and ción/European Development Regional Fund [FEDER], EU), (cofounded with FEDER Methods. funds) from the Spanish Ministerio de Economía, Industria y Competitividad.

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