Materials and Methods

Killer Archaea: Virus-Mediated Antagonism to CRISPR-Immune Populations Results in Emergent Virus-Host Mutualism

MATERIALS AND METHODSMicrobial strains. All Sulfolobus islandicus strains (see Table S1 in the supplemental material) were grown in dextrin-tryptone medium supplemented with uracil (DTU), as needed (29, 34). Cultures were grown at 75°C without shaking. Viral stocks and supernatants were collected at exponential growth of a chronically infected strain, filtered through a 0.2-μm polyethersulfone (PES) bottle-top filter (Nalgene catalog number 595-4520), and then stored at 4°C.Infections with SSV viruses. An S. islandicus immune-deficient ΔCas6 deletion strain was challenged with viruses as described previously (17). Following the 5-h incubation with virus filtrate, cells were resuspended in 70 ml DTU in a 75-cm2 culture flask and incubated at 75°C. Host growth was monitored, and after 8 days or when an optical density at 600 nm (OD600) of 0.10 was reached, cultures were plated in DTU-Gelrite overlays for colony isolation. To isolate a chronically infected strain after 10 to 14 days of incubation at 75°C, colonies were picked and resuspended in DTU and tested for infection using 16S rRNA- and SSV-specific primers. Colonies that tested positive for infection were then grown, supernatants were collected, and 10 μl was spotted on a lawn of the strain S. islandicus Y08.82.36 for production of PFU.Library prep and sequencing. Genomic libraries were prepared for all viruses using the Nextera XT kit (Illumina) according to the manufacturer's instructions. Libraries were pooled and sequenced using paired-end MiSeq version 2.5 by the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign. Reads were quality filtered using FASTX-Toolkit, and adapters were trimmed using Cutadapt (35). Host genome assemblies were compared to those of host strain M.16.4 using breseq (36).Determining SSV integration status. Quality filtered reads from a chronically infected strain were mapped to the SSV9 integrase by using bwa (37). Read names were extracted, and the mate pairs of mapped reads were then pulled into a separate file. Read mates were mapped back to the S. islandicus M.16.4 genome using bwa, and the alignment was visualized using Geneious. Areas of read coverage of the mate pairs were analyzed, and we found no evidence of reads mapping to the host genome above our map quality score threshold.Growth curves. To compare levels of growth of the uninfected and infected strains, cells were collected by centrifugation at 4,000 × g for 20 min. Cells were resuspended in fresh DTU and then diluted to a final volume of 20 ml at an initial OD600 of 0.04 and incubated without shaking at 75C. Cell growth was determined by measuring optical density every 24 h. At the time of sampling, 500-μl aliquots were taken and culture supernatants were collected by centrifugation at 15,000 × g in a microcentrifuge. Collected supernatants were then used for a plaque-forming assay. One hundred microliters of the supernatant was mixed with 500 μl of a 10×-concentrated strain, S. islandicus Y08.82.36. This mixture was incubated at 75°C for 30 min, and then 5 ml of an overlay sucrose-yeast (SY) medium was poured on SY plates as previously described (18). Plates were incubated at 75°C for 48 h.Transfer competitions. Strains used in competitions were grown to mid-log phase (OD600 of 0.10 to 0.18). Cells were collected by centrifugation at 4,000 × g for 20 min and resuspended in fresh media. Cells were then inoculated at the respective ratio of each type so that there was approximately 3 × 109 combined total cells. Cultures were incubated for 2 days at 75°C without shaking; after 2 days, the culture was diluted 1:5 into fresh medium. This was repeated 2 more times, for a total competition duration of 6 days. At the start of the competition and before and after dilution, 200 μl of culture was collected and frozen for analysis of cell abundance by qPCR and 200 μl of supernatant was collected for analysis of viral abundance (see Table S3 for primers). Primers targeting a 150-bp portion within the lacS gene or the residues left after lacS deletion were used to distinguish cell types using a previously described qPCR protocol. Standard curves were created using extracted genomic DNA from purified strains with and without the lacS gene. In the long-term transfer competitions, the experiment was conducted in a similar manner, but cells were transferred every 3 days.Nontransfer competitions. Strains used in competitions were grown to mid-log phase (OD600, 0.10 to 0.18). Cells were collected by centrifugation at 4,000 × g for 20 min and resuspended in fresh media. Cells were then inoculated at the respective ratio of each type so that there were approximately 3 × 109 total combined cells. Cultures were incubated at 75°C for 4 days, and 200 μl of culture was collected every day and frozen for analysis of cell abundance by qPCR as described above.Heat-killed supernatant assays. Cultures of the chronically infected strain or the isogenic uninfected strain were grown to an optical density of 0.15 to 0.20 and then filtered through a 0.22-μm PES filter. Supernatants were then placed in boiling water for 30 min. Supernatant samples from before and after boiling were tested for infectious particles by plaque assay as described above. When pepsin was used, the treatment of the supernatants was completed with immobilized pepsin (Thermo Scientific catalog number 20343) according to the manufacturer’s instructions and incubated at 37°C overnight in a rotator. Strains tested were grown to mid-log phase and then diluted to an OD600 of 0.03 in fresh medium, the boiled supernatants, or the unboiled supernatants. At 24, 48, and 72 h, cultures were centrifuged, and cells were resuspended in fresh doses of medium or the supernatants. Every 24 h, cell viability was tested by spotting 10-μl cell dilutions onto DTU plates.TABLE S2Primers used in this study. Download Table S2, DOCX file, 0.01 MB.Copyright © 2020 DeWerff et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Article TitleKiller Archaea: Virus-Mediated Antagonism to CRISPR-Immune Populations Results in Emergent Virus-Host Mutualism

Abstract

Theory, simulation, and experimental evolution demonstrate that diversified CRISPR-Cas immunity to lytic viruses can lead to stochastic virus extinction due to a limited number of susceptible hosts available to each potential new protospacer escape mutation. Under such conditions, theory predicts that to evade extinction, viruses evolve toward decreased virulence and promote vertical transmission and persistence in infected hosts. To better understand the evolution of host-virus interactions in microbial populations with active CRISPR-Cas immunity, we studied the interaction between CRISPR-immuneSulfolobus islandicuscells and immune-deficient strains that are infected by the chronic virus SSV9. We demonstrate thatSulfolobus islandicuscells infected with SSV9, and with other related SSVs, kill uninfected, immune strains through an antagonistic mechanism that is a protein and is independent of infectious virus. Cells that are infected with SSV9 are protected from killing and persist in the population. We hypothesize that this infection acts as a form of mutualism between the host and the virus by removing competitors in the population and ensuring continued vertical transmission of the virus within populations with diversified CRISPR-Cas immunity.


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