Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO2-derived 13C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of 13C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of 13C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in 13C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.
Microbially mediated oxidation of ammonia to nitrate during nitrification is a central component of the global nitrogen (N) cycle. It is also responsible for major losses of applied fertiliser N in soil, generating atmospheric pollution via direct and indirect production of nitrous oxide (N2O) as well as nitrate (NO3-) pollution of groundwater . Autotrophic ammonia-oxidising archaea (AOA) of the class Nitrososphaeria are a ubiquitous component of soil microbial communities and often dominate ammonia oxidation and nitrification-associated N2O emissions when ammonia is supplied at low rates via organic matter mineralisation , slow-release fertilisers  or in acidic soils . Integrated temperate viruses (proviruses) and other virus-associated protein-encoding genes are found in most AOA genomes suggesting frequent interaction (see Supplementary Text). While viruses infecting marine AOA have been characterised through metagenomic approaches  and cultivation , those infecting soil AOA or other nitrifier groups are currently uncharacterised.
Virus infection can influence biogeochemical cycling by augmenting host activity or causing cell mortality and subsequent release of nutrients . Recent advances have demonstrated that soil virus communities are dynamic in a wide range of soils [e.g. 8, 9] and augmenting virus loads modulate C and N fluxes [10, 11]. Nevertheless, identifying active interactions with specific populations or functional groups in soil remains challenging due to structural complexity and the vast diversity of hosts and viruses. Recent use of stable-isotope approaches has investigated whole community host-virus dynamics [12, 13] or interactions between individual host-virus populations specific to a functional process and substrate . The aim of this study was to utilise the latter approach with 13CO2-based DNA-SIP to focus on nitrification-associated interactions and to test the hypothesis that viruses are a dynamic component of soil AOA activity.
Hybrid analysis of GC mol% fractionation and 13C-DNA-SIP for identifying active AOA hosts and viruses
Microcosms were established (see Supplementary Text) with two sandy loam soils of contrasting pH (4.5 or 7.5), which were characterised previously as containing distinct AOA communities . As ammonia from hydrolysed urea is used by AOA in these and other soils [1, 3], nitrification was fuelled by addition of urea (two pulses of 100 µg N g−1 soil (dry weight)) to stimulate AOA growth and potential enrichment of AOA-infecting viruses. Microcosms were incubated at 25 oC for 30 days (Supplementary Fig. 1) with a 5% 12C- or 13C-CO2 headspace to target viruses of autotrophs via transfer of assimilated carbon. Extracted DNA was subjected to isopycnic centrifugation in CsCl gradients, in which DNA migrates as a function of GC mol% and isotopic enrichment, before fractionation and analysis of genomic DNA of different buoyant densities . Quantification of AOA genomic DNA distribution across CsCl gradients demonstrated growth of AOA populations (Fig. 1A; Supplementary Fig. 2) and genomic DNA in high buoyant density (HBD) fractions (>1.719 g ml−1) was then pooled for each replicate microcosm and metagenomes sequenced (Supplementary Table 1). While 13C-incorporation clearly separated DNA from growing vs non-growing AOA populations, 13C-enriched AOA genomes (average contig GC mol% content of 38%) co-migrated within the CsCl gradient with more abundant community 12C genomes with higher GC mol% (>57%), reducing AOA host and virus sequencing depth, contig assembly and binning. Consequently, only one medium-quality AOA metagenome-assembled genome (MAG) was recovered from HBD DNA and no AOA-associated virus contigs were identified amongst 290 viral operational taxonomic units (vOTUs) ≥10 kb (Fig. 1B).
To facilitate analysis of low GC mol% genomes while also identifying activity, we developed a hybrid approach combining metagenomic analysis of low buoyant density (LBD) 12C DNA with read-mapping of sequenced HBD DNA (Fig. 1; Supplementary Fig. 2). The AOA and virus genomes were assembled from metagenomic libraries of 12C LBD DNA only (<1.699 g ml−1) representing both dormant and active populations. Individual sequence reads from HBD DNA libraries from both 12C- and 13C-incubations were then mapped to identify activity with a significantly greater relative abundance in 13C-incubated samples demonstrating isotope incorporation. For taxonomically defined contigs of hosts or host-linked viruses, in comparison to HBD, assemblies from LBD libraries increased the relative proportion of contigs from the AOA-containing Thermoproteota phylum (GTDB taxonomy; Nitrososphaerota in ICNP taxonomy and Thaumarchaeota in NCBI taxonomy) and Thermoproteota-infecting virus contigs from 0.3 to 2.8% and 0 to 22.3%, respectively (Supplementary Fig. 3). In LBD metagenomes, 123 medium- and high-quality MAGs from both soils were assigned taxonomy (Supplementary Table 2) including nine AOA MAGs representing six genera (GTDB classification ) within the Nitrososphaerales and Nitrosopumilales orders and one nitrite-oxidising bacterium (NOB) Nitrobacter MAG (MAG 15) (Fig. 1B; Supplementary Fig. 4), contributing to the oxidation of nitrite (NO2-) to NO3- during the second step of canonical nitrification. Mapping of HBD sequence reads revealed that eight of the ten MAGs were derived from active autotrophic populations (Fig. 1C). Consistent with previous studies characterising the distribution of soil AOA from different taxa , this included four Nitrosotalea genus populations (MAGs 9, 61, 77 and 107) from pH 4.5 soil and three Nitrososphaera genus populations (MAGs 12, 52 and 96) from pH 7.5 soil. No evidence of autotrophic growth was observed for populations represented by a further two MAGs within the Nitrososphaerales. Both are from genera with no cultivated representatives with MAG 112 from genus TA-21 (amoA-gene lineage NS-β-2 ) and MAG 63 (amoA-gene lineage NS-δ-2.1) representing a novel genus (designated here as CS63). The latter belongs to the amoA gene-defined NS-δ lineage that while widely found in soil, is rarely implicated in contributing to ammonia oxidation. Although these two MAGs may simply be from AOA populations not active under the incubation conditions used, they are also consistent with microorganisms possessing physiologies distinct from cultivated AOA.
After predicting contigs of virus origin using established bioinformatic tools, those from AOA-infecting viruses were identified with a custom database of hallmark genes from AOA proviruses and analysis of other shared homologues (see Supplementary Text). Specifically, proviruses were identified in 184 (87%) of 212 AOA genome sequences downloaded from GTDB. Capsid, terminase, portal and integrase protein sequences from these proviruses were subsequently used as search queries. While previous analysis of 12 total soil metagenome or virome libraries from the same soil samples did not enable identification of any AOA-associated virus contigs (average of 152 million quality filtered reads and 1,947 contigs ≥10 kb per library) , this targeted approach using LBD DNA from nitrifying microcosms yielded 64 contigs predicted as derived from AOA-infecting viruses and each representing a unique vOTU. In addition, viruses infecting other nitrifier populations were identified with five and three vOTUs derived from viruses infecting ammonia-oxidising bacteria (AOB) and NOB, respectively. Virus and host genome GC mol% correlate  and AOA-infecting virus contigs possessed a similar average content of 36.3% which was considerably lower than averages of 46.6% and 67.0% of all viral contigs recovered from LBD and HBD metagenomes, respectively. As with their hosts, AOA virus communities were distinct between the two pH soils (Fig. 2A) as also observed previously for total virus communities  with 29 (45%) and 34 (53%) vOTUs significantly more relatively abundant in pH 4.5 and 7.5 LBD libraries compared to the other soil, respectively. Comparison of the relative abundance of HDB sequence reads from both 12C and 13C libraries revealed that 27 contigs from both soils (42%) were enriched in 13C and thus confirming infection during incubation and transfer of C from AOA hosts to viruses.
Soil AOA viruses were distinct from characterised archaeal and bacterial viruses. Gene-sharing network analysis using vConTACT v2.0  did not identify any associations with reference sequences or recently described viruses of marine AOA  and genome-wide sequence comparisons  placed 60 of the 64 soil AOA viral contigs within a unique proteomic grouping (Fig. 2B). The majority were predicted as capable of lysogeny with 55 encoding an integrase. While it can be difficult to determine virulent versus temperate capability from partial genomes, only 6.8 and 8.9% of all virus contigs from LBD and HBD metagenomes, respectively, possessed a predicted integrase. Although suggesting a higher proportion of AOA viruses may be temperate, it should be noted that this could be a consequence of using AOA provirus-associated genes to identify viral contigs, biasing towards identification of those from temperate viruses. These results also highlight that while DNA-SIP of total metagenomes enables identification of 13C incorporation into virus genomes, it is potentially limited in facilitating confident bioinformatic prediction of free temperate virus particles versus proviruses present from a previous infection of a currently active host. While the integration status of a temperate virus can be predicted bioinformatically, using targeted “viromes” metagenomes of filtered virus particles will provide greater confidence of identifying genomes from free virus particles.
Eleven contigs contained large terminase (TerL) sub-unit gene sequences and were similar to those recovered from other predicted AOA virus or provirus genomes only (e-value <10−5) (Fig. 2C). In addition to viral hallmark genes encoding portal, endonuclease, tail and capsid proteins, contig 053596 contained a type 1 multicopper oxidase (MCO1) homologue found exclusively in AOA genomes  (Fig. 2D). Copper (Cu) is essential for ammonia oxidation and electron transport in AOA and MCO1 is upregulated under Cu limitation and potentially involved in Cu sequestration from the environment . This virus-encoded MCO1 homologue therefore represents an auxiliary metabolic gene that may contribute to a core process in AOA physiology.
These results from two soils indicate that virus infection of AOA during nitrification is likely a common and dynamic process, with the potential to influence nitrification rates in soil. Although viruses were only identified from contigs representing partial viral genomes, analysis of individual marker genes and genome-wide similarity comparisons revealed that those infecting AOA are highly divergent from known viruses. While one Nitrobacter MAG was obtained and both AOB and NOB viruses were also predicted, the hybrid approach developed in this study targeted low GC mol% DNA and was specifically tailored for the analysis of AOA populations and their viruses. A broader analysis of all viruses infecting nitrifying prokaryotic populations would therefore likely require a more comprehensive sequencing effort of all fractions throughout the CsCl gradient using quantitative SIP metagenomics  or alternative approaches, such as virus-targeted metagenomic viromes.
Metagenome sequence data are deposited in NCBI’s GenBank under BioProject accession nos. PRJNA621418-PRJNA621429 (13C data) and PRJNA868779 (12C data) with MAGs available under accession numbers SAMN30436125- SAMN30436246.
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This work was funded by an AXA Research Chair in Microbial Ecology awarded to GWN, Agence Nationale de la Recherche grant ‘CONSERVE’ (ANR-22-CE02-0006-01) awarded to GWN and CH, and an EU Horizon Europe MSCA Postdoctoral Fellowship ‘DIVOBIS’ (101064472) awarded to ETS. Sequencing data generated under JGI Community Science Program Proposal No. 503702 awarded to GWN and CH was conducted by the US DOE JGI, a DOE Office of Science User Facility, and supported by the Office of Science of the US DOE under Contract No. DE-AC02-05CH11231. We acknowledge use of the Newton high-performance computing cluster at PMCS2I, École Centrale de Lyon. The authors would like to thank Dr. Willm Martens-Habbena (University of Florida) for continued discussions on soil AOA ecophysiology and taxonomy, Dr. Melina Kerou (University of Vienna) for guidance on AOA MCO diversity, Dr. Gary Trubl for comments on the manuscript and Dr. Robin Walker (SRUC, Aberdeen) for access to the much-lamented Craibstone Estate Woodlands Field long term experiment.
The authors declare no competing interests.
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Lee, S., Sieradzki, E.T., Nicol, G.W. et al. Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification. ISME J 17, 309–314 (2023). https://doi.org/10.1038/s41396-022-01341-5