Abstract
It is generally assumed that viruses outnumber cells on Earth by at least tenfold. Virus-to-microbe ratios (VMR) are largely based on counts of fluorescently labelled virus-like particles. However, these exclude intracellular viruses and potentially include false positives (DNA-containing vesicles, gene-transfer agents, unspecifically stained inert particles). Here, we develop a metagenome-based VMR estimate (mVRM) that accounts for DNA viruses across all stages of their replication cycles (virion, intracellular lytic and lysogenic) by using normalised RPKM (reads per kilobase of gene sequence per million of mapped metagenome reads) counts of the major capsid protein (MCP) genes and cellular universal single-copy genes (USCGs) as proxies for virus and cell counts, respectively. After benchmarking this strategy using mock metagenomes with increasing VMR, we inferred mVMR across different biomes. To properly estimate mVMR in aquatic ecosystems, we generated metagenomes from co-occurring cellular and viral fractions (>50 kDa–200 µm size-range) in freshwater, seawater and solar saltern ponds (10 metagenomes, 2 control metaviromes). Viruses outnumbered cells in freshwater by ~13 fold and in plankton from marine and saline waters by ~2–4 fold. However, across an additional set of 121 diverse non-aquatic metagenomes including microbial mats, microbialites, soils, freshwater and marine sediments and metazoan-associated microbiomes, viruses, on average, outnumbered cells by barely two-fold. Although viruses likely are the most diverse biological entities on Earth, their global numbers might be closer to those of cells than previously estimated.
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Data availability
Metagenome Illumina sequences generated in our laboratory and not yet published have been deposited in GenBank (National Centre for Biotechnology Information) Short Read Archive with BioProject number PRJNA756245. Accession numbers for all used metagenomes are provided in Table S8. Metagenome assemblies generated for this study are available here: https://www.deemteam.fr/en/datasets. The corresponding proteomes as well as the HMM profiles are available in FigShare (https://figshare.com/projects/mVMRs/159701) and all code is available in GitLab (https://gitlab.com/anagtz/mvmrs).
Code availability
Metagenome Illumina sequences generated in our laboratory and not yet published have been deposited in GenBank (National Centre for Biotechnology Information) Short Read Archive with BioProject number PRJNA756245. Accession numbers for all used metagenomes are provided in Table S8. Metagenome assemblies generated for this study are available here: https://www.deemteam.fr/en/datasets. The corresponding proteomes as well as the HMM profiles are available in FigShare (https://figshare.com/projects/mVMRs/159701) and all code is available in GitLab (https://gitlab.com/anagtz/mvmrs).
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Acknowledgements
We thank José Manuel Haro Moreno for help during marine and solar saltern sampling in Alicante, Paola Bertolino for help during sampling in several other ecosystems, and Antonio Camacho y Antonio Picazo for CTD measurements during marine plankton sampling. We thank the Salinas Bras del Port for sampling permission and the UNICELL platform for flow cytometry analyses. We acknowledge support from the Moore-Simons Project on the Origin of the Eukaryotic Cell through Grant GBMF9739 (PL-G) and the European Research Council Advanced Grants FP7 No. 322669 (PL-G) and H2020 No. 787904 (DM).
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PL-G and DM conceived the study and designed experiments, organised and participated in sampling and successive filtration steps, did the tangential filtration and quantitatively purified DNA from the different cell and viral fractions. AG-P participated in sampling and carried out all bioinformatic analyses, with informatics support by PD. MK designed the HMM profiles for MCPs. MC carried out flow cytometry counts of viruses and cells with the assistance of LJ. ML-P and FR-V co-organised sampling in marine and solar saltern sites and helped with filtration on board. PL-G wrote the manuscript with input from AG-P and MC. All authors read, commented and approved the manuscript.
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López-García, P., Gutiérrez-Preciado, A., Krupovic, M. et al. Metagenome-derived virus-microbe ratios across ecosystems. ISME J 17, 1552–1563 (2023). https://doi.org/10.1038/s41396-023-01431-y
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DOI: https://doi.org/10.1038/s41396-023-01431-y