Lytic archaeal viruses infect abundant primary producers in Earth's crust

Abstract

The continental subsurface houses a major portion of life's abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we use a combination of metagenomics and virus-targeted direct-geneFISH (virusFISH) to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches display resistances of Ca. Altiarchaea against eight predicted viral clades, which show genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tag and image a putatively viral genome rich in protospacers using fluorescence microscopy. VirusFISH reveals a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.

Fig. 1. Global distribution of abundant Altiarchaeota (group Alti-1) and their predicted viruses in Altiarchaeota hot spots.

Distribution analysis is based on 16S rRNA gene sequencing (yellow dots) and the detection of hamus genes in metagenomes from IMG (blue and purple dots). Purple dots correspond to the four investigated sites of this study. Normalized host and virus coverage are given for the four subsurface habitats: Alpena County Library Fountain (ACLF, Michigan, USA), Horonobe Underground Research Laboratory (HURL, Japan), Mühlbacher Schwefelquelle, Isling (MSI, Germany), and Geyser Andernach (GA, Germany). Percent relative abundance of dominant Altiarchaeota compared to other community members is shown in Supplementary Table 1. Only Altivir_1_MSI and Altivir_2_MSI obtained from biofilm (BF) samples are shown. The letter C indicates circular genomes. n.d. none detected. World map has been generated using Ocean Data View v.5.3.0. Color explanation of bars representing normalized coverage: orange-red = Altivir_1_MSI (virus), blue = Altivir_2_MSI, purple = Altivir_3_ACLF, rose = Altivir_4_ACLF, brown = Altivir_5_ACLF, light blue = Altivir_6_ACLF, green = Altivir_7_ACLF, and black = Altiarchaeota (host).

Fig. 2. Phylogenomic genome-BLAST distance phylogeny (GBDP) tree of Ca. Altiarchaeum viruses and viral proteins clusters.

a The tree was inferred using the distance formula D0 yielding average support of 69%. The numbers above branches are GBDP pseudo-bootstrap support values from 100 replications. The branch lengths of the resulting VICTOR trees are scaled in terms of the respective distance formula used. The tree shows that the eight predicted viral genomes were assigned to the same family, to eight different genera, and ten species (which is the result of having multiple genomes included for Altivir_1_MSI and Altivir_2_MSI). b Protein clustering across all viruses revealed that Altivir_1_MSI_>0.1 µm_2018 and Altivir_6_ACLF, originating from different continents, have five protein clusters (2–6) in common. Altivir_2_MSI_BF_2012, Altivir_4_ACLF, and Altivir_8_HURL shared only one protein cluster (1). Open reading frames of the viral genomes were predicted using Prodigal v.2.6.3 and further translated with the R package seqinr v.3.6.-1. Colors indicate shared protein clusters between genomes and numbers show nonhypothetical consensus annotations according to Supplementary Data 3. Color code for viral genomes: Altivir_1_MSI = black, Altivir_2_MSI = blue, Altivir_3_ACLF = yellow, Altivir_4_ACLF = pink, Altivir_5_ACLF = orange, Altivir_6_ACLF = olive-green, Altivir_7_ACLF = pink-red, and Altivir_8_HURL = purple.

Fig. 3. Visualization and quantification of Ca. Altiarchaeum virus Altivir_1_MSI-infected and noninfected Altiarchaeota biofilm (BF) cells from MSI site.

For all virusFISH experiments shown here, an Altivir_1_MSI probe was used for detecting viral infections. BF material was visualized with filter sets for DAPI (blue, cells), ATTO 488 (purple, 16S rRNA signal), and Alexa 594 (yellow, viral genomes), and merged for analysis and display purposes. a VirusFISH displays the interactions between Altiarchaeota cells and their virus shown as yellow dots. For unmerged imaging data, see Supplementary Fig. 11. Scale bar: 10 µm. b Different infection stages with Altivir_1_MSI. The enumeration performed with a regular epifluorescence microscope was based on 18,411 archaeal cells and categorized into three infection stages. For the categorization we used in total 18 Altiarchaeota biofilms, whereby 17 biofilms were treated with the Altivir_1_MSI probe (n = 17) and one biofilm was treated with a Metallosphaera sp. virus probe (n = 1). Purple arrows indicate exemplary viruses that attach to host’s cell surface, white arrows show advanced infections, and orange arrows bursting cells with free viruses. Scale bars: 2 µm. c Coupling virusFISH with structured illumination microscopy showed extracellular signals of tiny fluorescently labeled viral particles attaching to Altiarchaeota’s cell surface but also in a free state as presumably released virions (n = 1). Scale bar: 2 µm. d Transmission electron microscopy revealed intracellular virus-like particles (n = 3). Scale bar: 100 nm.

Fig. 4. Development of host–virus and spacer dynamics from 2012 to 2018 based on metagenomics from the Mühlbacher Schwefelquelle, Isling (MSI).

Predicted viruses Altivir_1_MSI and Altivir_2_MSI became less abundant from 2012 to 2018. Data are presented for biofilms (BF) from 2012 and 2018 as well as the planktonic fraction (>0.1 µm) and the viral fraction (<0.1 µm). Considering the BFs, total spacer abundance and numbers increased from 2012 to 2018 while those matching Altivir_1_MSI decreased in abundance but increased in numbers. Number (no.) of microbial taxa refers to the number of different prokaryotes in a sample detected via rpS3 rank abundance curves (normalized by sequencing depth). Host–virus ratio is calculated from host and virus coverage based on read mapping. Abundance and number of different spacers were normalized to minimum relative abundance (rel. abd.) of the host based on read mapping. Color definition of bars: violet = number of microbial taxa, orange-red = features of Altivir_1_MSI, blue = features of Altivir_2_MSI, black = log₁₀ relative abundance of Altiarchaeum, ocher = number of all spacers, and purple = abundance of all spacers.

Fig. 5. VirusFISH of two individual Altiarchaeota biofilm (BF) flocks depicting a) a dense BF flock without infections and b) one highly infected BF flock.

For all virusFISH experiments, an Altivir_1_MSI probe was used for detecting viral infections. White arrows indicate exemplary virus–Altiarchaeota interactions (advanced infections). BF material was analyzed with filter sets for DAPI (blue, cells), ATTO 488 (purple, 16S rRNA signal), and Alexa 594 (yellow, viral genomes), and then the different fluorescent channels were merged for analysis and display purposes. For unmerged imaging data, see Supplementary Figs. 16 and 17. Scale bars: 10 µm.