Spindle-shaped viruses infect marine ammonia- 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 Microbiology, 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 archaea (AOA) from the phylum Thaumarchaeota viruses of the order Caudovirales have been previously identified in are ubiquitous in marine ecosystems and play a prominent role in the genomes 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-cell genomic studies have resulted in the as- mortality, thereby regulating global biogeochemical cycles. However, sembly of putative AOA virus 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 Nitrosopumilus spindle-shaped viruses (NSVs) that infect ammonia monooxygenase subunit C (amoC), a key component of AOA and are distinct from other known marine viruses. Although ammonia monooxygenase (AMO) (13, 14), suggesting an active role NSVs have a narrow host 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 Bacteria 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 bacteriophages, 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, evolution, 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, species, 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 genome 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 www.pnas.org/cgi/doi/10.1073/pnas.1905682116 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 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 coral 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 life 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 genus 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