The deep terrestrial biosphere harbours a substantial fraction of Earth’s biomass and remains understudied compared with other ecosystems. Deep biosphere life primarily consists of bacteria and archaea, yet knowledge of their co-occurring viruses is poor. Here, we temporally catalogued viral diversity from five deep terrestrial subsurface locations (hydraulically fractured wells), examined virus–host interaction dynamics and experimentally assessed metabolites from cell lysis to better understand viral roles in this ecosystem. We uncovered high viral diversity, rivalling that of peatland soil ecosystems, despite low host diversity. Many viral operational taxonomic units were predicted to infect Halanaerobium, the dominant microorganism in these ecosystems. Examination of clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins (CRISPR–Cas) spacers elucidated lineage-specific virus–host dynamics suggesting active in situ viral predation of Halanaerobium. These dynamics indicate repeated viral encounters and changing viral host range across temporally and geographically distinct shale formations. Laboratory experiments showed that prophage-induced Halanaerobium lysis releases intracellular metabolites that can sustain key fermentative metabolisms, supporting the persistence of microorganisms in this ecosystem. Together, these findings suggest that diverse and active viral populations play critical roles in driving strain-level microbial community development and resource turnover within this deep terrestrial subsurface ecosystem.
Subscribe to Journal
Get full journal access for 1 year
only $5.17 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Halanaerobium isolate genome and MAGs are publicly available in the JGI Genome Portal database (http://img.jgi.doe.gov/) or in NCBI; see Supplementary Table 1 for accession numbers. All of the metagenomic nucleotide files used in this study are publicly available through JGI or NCBI; accession numbers are listed in Supplementary Data 1.
Suttle, C. A. Viruses in the sea. Nature 437, 356–361 (2005).
Whitman, W. B., Coleman, D. C. & Wiebe, W. J. Prokaryotes: the unseen majority. Proc. Natl Acad. Sci. USA 95, 6578–6583 (1998).
De Smet, J. et al. High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection. ISME J. 10, 1823–1835 (2016).
Wommack, K. E. & Colwell, R. R. Virioplankton: viruses in aquatic ecosystems. Microbiol. Mol. Biol. Rev. 64, 69–114 (2000).
De Smet, J., Hendrix, H., Blasdel, B. G., Danis-Wlodarczyk, K. & Lavigne, R. Pseudomonas predators: understanding and exploiting phage–host interactions. Nat. Rev. Microbiol. 15, 517–530 (2017).
Feiner, R. et al. A new perspective on lysogeny: prophages as active regulatory switches of bacteria. Nat. Rev. Microbiol. 13, 641–650 (2015).
Brum, J. R. et al. Patterns and ecological drivers of ocean viral communities. Science 348, 1261498–1261498 (2015).
Danovaro, R. et al. Major viral impact on the functioning of benthic deep-sea ecosystems. Nature 454, 1084–1087 (2008).
Emerson, J. B. et al. Host-linked soil viral ecology along a permafrost thaw gradient. Nat. Microbiol. 3, 870–880 (2018).
Nigro, O. D. et al. Viruses in the oceanic basement. mBio 8, e02129-16 (2017).
Pan, D. et al. Correlation between viral production and carbon mineralization under nitrate-reducing conditions in aquifer sediment. ISME J. 8, 1691–1703 (2014).
Reyes, A. et al. Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466, 334–338 (2010).
Anderson, R. E., Brazelton, W. J. & Baross, J. A. Is the genetic landscape of the deep subsurface biosphere affected by viruses? Front. Microbiol. 2, 219 (2011).
Daly, R. A. et al. Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales. Nat. Microbiol. 1, 16146 (2016).
Booker, A. E. et al. Sulfide generation by dominant Halanaerobium microorganisms in hydraulically fractured shales. mSphere 2, e00257-17 (2017).
Liang, R. et al. Metabolic capability of a predominant Halanaerobium sp. in hydraulically fractured gas wells and its implication in pipeline corrosion. Front. Microbiol. 7, 116 (2016).
Lipus, D. et al. Predominance and metabolic potential of Halanaerobium in produced water from hydraulically fractured Marcellus shale wells. Appl. Environ. Microbiol. 83, 181 (2017).
Mouser, P. J., Borton, M., Darrah, T. H., Hartsock, A. & Wrighton, K. C. Hydraulic fracturing offers view of microbial life in the deep terrestrial subsurface. FEMS Microbiol. Ecol. 92, fiw166 (2016).
Bhupathiraju, V. K., McInerney, M. J., Woese, C. R. & Tanner, R. S. Haloanaerobium kushneri sp nov., an obligately halophilic, anaerobic bacterium from an oil brine. Int. J. Syst. Evol. Microbiol. 49, 953–960 (1999).
Bhupathiraju, V. K. et al. Haloanaerobium salsugo Sp-Nov, a moderately halophilic, anaerobic bacterium from a subterranean brine. Int. J. Syst. Evol. Microbiol. 44, 565–572 (1994).
Brown, S. D. et al. Complete genome sequence of the haloalkaliphilic, hydrogen-producing bacterium Halanaerobium hydrogeniformans. J. Bacteriol. 193, 3682–3683 (2011).
Kivisto, A. et al. Genome sequence of Halanaerobium saccharolyticum subsp. saccharolyticum strain DSM 6643T, a halophilic hydrogen-producing bacterium. Genome Announc. 1, e00187-13 (2013).
Zeikus, J. G., Hegge, P. W., Thompson, T. E., Phelps, T. J. & Langworthy, T. A. Isolation and description of Haloanaerobium praevalens gen. nov. and sp. nov., an obligately anaerobic halophile common to Great Salt Lake sediments. Curr. Microbiol. 9, 225–233 (1983).
Roux, S., Hallam, S. J., Woyke, T. & Sullivan, M. B. Viral dark matter and virus–host interactions resolved from publicly available microbial genomes. eLife 4, e08490 (2015).
Gregory, A. C. et al. Genomic differentiation among wild cyanophages despite widespread horizontal gene transfer. BMC Genomics 17, 930 (2016).
Leff, J. W. et al. Consistent responses of soil microbial communities to elevated nutrient inputs in grasslands across the globe. Proc. Natl Acad. Sci. USA 112, 10967–10972 (2015).
Bolduc, B. et al. vConTACT: an iVirus tool to classify double-stranded DNA viruses that infect Archaea and Bacteria. PeerJ 5, e3243 (2017).
Roux, S. et al. Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses. Nature 537, 689–693 (2016).
Paez-Espino, D. et al. Uncovering Earth’s virome. Nature 536, 425–430 (2016).
Lefkowitz, E. J. et al. Virus taxonomy: the database of the International Committee on Taxonomy of Viruses (ICTV). Nucleic Acids Res. 46, D708–D717 (2018).
Cluff, M. A., Hartsock, A., MacRae, J. D., Carter, K. & Mouser, P. J. Temporal changes in microbial ecology and geochemistry in produced water from hydraulically fractured Marcellus shale gas wells. Environ. Sci. Technol. 48, 6508–6517 (2014).
Davis, J. P., Struchtemeyer, C. G. & Elshahed, M. S. Bacterial communities associated with production facilities of two newly drilled thermogenic natural gas wells in the Barnett Shale (Texas, USA). Microb. Ecol. 64, 942–954 (2012).
Murali Mohan, A. et al. Microbial community changes in hydraulic fracturing fluids and produced water from shale gas extraction. Environ. Sci. Technol. 47, 13141–13150 (2013).
Wuchter, C., Banning, E. & Mincer, T. J. Microbial diversity and methanogenic activity of Antrim Shale formation waters from recently fractured wells. Front. Microbiol. 4, 367 (2013).
Ahlgren, N. A., Ren, J., Lu, Y. Y., Fuhrman, J. A. & Sun, F. Alignment-free d 2 * oligonucleotide frequency dissimilarity measure improves prediction of hosts from metagenomically-derived viral sequences. Nucleic Acids Res. 45, 39–53 (2017).
Wang, K., Wommack, K. E. & Chen, F. Abundance and distribution of Synechococcus spp. and cyanophages in the Chesapeake Bay. Appl. Environ. Microbiol. 77, 7459–7468 (2011).
Sharon, I. et al. Time series community genomics analysis reveals rapid shifts in bacterial species, strains, and phage during infant gut colonization. Genome Res. 23, 111–120 (2013).
Waterbury, J. B. & Valois, F. W. Resistance to co-occurring phages enables marine Synechococcus communities to coexist with cyanophages abundant in seawater. Appl. Environ. Microbiol. 59, 3393–3399 (1993).
Stern, A. & Sorek, R. The phage-host arms race: shaping the evolution of microbes. Bioessays 33, 43–51 (2010).
Howard-Varona, C. et al. Multiple mechanisms drive phage infection efficiency in nearly identical hosts. ISME J. 12, 1605–1618 (2018).
Sun, C. L., Thomas, B. C., Barrangou, R. & Banfield, J. F. Metagenomic reconstructions of bacterial CRISPR loci constrain population histories. ISME J. 10, 858–870 (2015).
Emerson, J. B. et al. Virus–host and CRISPR dynamics in archaea-dominated hypersaline Lake Tyrrell, Victoria, Australia. Archaea 2013, 1–12 (2013).
Achigar, R., Magadán, A. H., Tremblay, D. M., Pianzzola, M. J. & Moineau, S. Phage–host interactions in Streptococcus thermophilus: genome analysis of phages isolated in Uruguay and ectopic spacer acquisition in CRISPR array. Sci. Rep. 7, 43438 (2017).
Gómez, P. & Buckling, A. Bacteria–phage antagonistic coevolution in soil. Science 332, 106–109 (2011).
Levin, B. R. Nasty viruses, costly plasmids, population dynamics, and the conditions for establishing and maintaining CRISPR-mediated adaptive immunity in bacteria. PLoS Genet. 6, e1001171 (2010).
Brum, J. R., Schenck, R. O. & Sullivan, M. B. Global morphological analysis of marine viruses shows minimal regional variation and dominance of non-tailed viruses. ISME J. 7, 1738–1751 (2013).
Kauffman, K. M. et al. A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria. Nature 554, 118–112 (2018).
Bondy-Denomy, J. et al. Prophages mediate defense against phage infection through diverse mechanisms. ISME J. 10, 2854–2866 (2016).
Borton, M. A. et al. Coupled laboratory and field investigations resolve microbial interactions that underpin persistence in hydraulically fractured shales. Proc. Natl Acad. Sci. USA 104, 201800155 (2018).
Weitz, J. S. et al. A multitrophic model to quantify the effects of marine viruses on microbial food webs and ecosystem processes. ISME J. 9, 1352–1364 (2015).
John, S. G. et al. A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environ. Microbiol. Rep. 3, 195–202 (2010).
Lever, M. A. et al. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types. Front. Microbiol. 6, 1281 (2015).
Joshi, N. A. & Fass, J. N. Sickle: A Sliding-Window, Adaptive, Quality-Based Trimming Tool for FastQ Files Version 1.33 (2011); https://github.com/najoshi/sickle
Wu, M. & Scott, A. J. Phylogenomic analysis of bacterial and archaeal sequences with AMPHORA2. Bioinformatics 28, 1033–1034 (2012).
Miller, C. S., Baker, B. J., Thomas, B. C., Singer, S. W. & Banfield, J. F. EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data. Genome Biol. 12, R44 (2011).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Leplae, R., Hebrant, A., Wodak, S. J. & Toussaint, A. ACLAME: a CLAssification of Mobile genetic Elements. Nucleic Acids Res. 32, D45–D49 (2004).
Shannon, P. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).
Sullivan, M. J., Petty, N. K. & Beatson, S. A. Easyfig: a genome comparison visualizer. Bioinformatics 27, 1009–1010 (2011).
Makarova, K. S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).
Weljie, A. M., Newton, J., Mercier, P., Carlson, E. & Slupsky, C. M. Targeted profiling: quantitative analysis of 1H NMR metabolomics data. Anal. Chem. 78, 4430–4442 (2006).
Thurber, R. V., Haynes, M., Breitbart, M., Wegley, L. & Rohwer, F. Laboratory procedures to generate viral metagenomes. Nat. Protoc. 4, 470–483 (2009).
Williamson, S. J., Houchin, L. A., McDaniel, L. & Paul, J. H. Seasonal variation in lysogeny as depicted by prophage induction in Tampa Bay, Florida. Appl. Environ. Microbiol. 68, 4307–4314 (2002).
Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).
R.A.D., M.A.B, D.M.M, A.E.B, A.J.H, P.J.M., K.C.W. and M.J.W. are partially supported by funding from the National Sciences Foundation Dimensions of Biodiversity (award no. 1342701). R.A.D., M.A.B., D.M.M., A.E.B., K.C.W. and M.J.W. also received support from Dow Microbial Control for this work. Samples from wells M-4 and M-5 were provided by the Marcellus Shale Energy and Environment Laboratory funded by the Department of Energy’s National Energy Technology Laboratory, grant no. DE-FE0024297. Metagenomic sequencing for this research was performed by the Department of Energy’s Joint Genome Institute (JGI) via a large-scale sequencing award to K.C.W (award no. 1931). Metabolite support was provided by Environmental Molecular Sciences Laboratory (EMSL) support via a JGI–EMSL Collaborative Science Initiative awarded to K.C.W (award no. 48483) and an EMSL instrument time award to M.J.W. (award no. 49615). Both JGI and EMSL facilities are sponsored by the Office of Biological and Environmental Research and operated under contract nos. DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL). M.B.S. was partially supported by a Gordon and Betty Moore Foundation grant (no. 3790).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Tables 1–4, Supplementary Figures 1–5.
Sequencing information for metagenomes.
Viral OTU table.
Halanaerobium relative abundance in the Utica-2 well.
Prophage induction metabolites.
About this article
Cite this article
Daly, R.A., Roux, S., Borton, M.A. et al. Viruses control dominant bacteria colonizing the terrestrial deep biosphere after hydraulic fracturing. Nat Microbiol 4, 352–361 (2019). https://doi.org/10.1038/s41564-018-0312-6
Environmental Microbiome (2020)
Genome-Resolved Metagenomics Extends the Environmental Distribution of the Verrucomicrobia Phylum to the Deep Terrestrial Subsurface
Viral and bacterial community responses to stimulated Fe(III)‐bioreduction during simulated subsurface bioremediation
Environmental Microbiology (2019)
Evaluation of Sequencing Library Preparation Protocols for Viral Metagenomic Analysis from Pristine Aquifer Groundwaters