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Pioneering Soil Viromics to Elucidate Viral Impacts on Soil Ecosystem Services

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Pioneering Soil Viromics to Elucidate Viral Impacts on Soil Ecosystem Services DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Gareth Trubl Graduate Program in Microbiology The Ohio State University 2018 Dissertation Committee: Virginia Rich, Advisor Matthew Sullivan, Co-Advisor Kelly Wrighton Matthew Anderson Copyrighted by Gareth Trubl 2018 Abstract Permafrost contains 30–50% of global soil carbon (C) and is rapidly thawing. While the fate of this C is unknown, it will be shaped in part by microbes and their associated viruses, which modulate microbial activities via mortality and metabolic control. To date, viral research in soils has been outpaced by that in aquatic environments due to the technical challenges of accessing soil viruses, compounded by the dramatic physicochemical heterogeneity in soils. The Stordalen Mire long-term ecological field site in Arctic Sweden encompasses a mosaic of natural permafrost thaw stages, and has been well characterized biogeochemically and microbiologically, making it an ideal site to characterize the soil virosphere and its potential impacts on the C cycle. A viral resuspension protocol was developed to generate quantitatively- amplified dsDNA viromes. The protocol yielded ~108 virus-like particles (VLPs) g−1 of soil across three thaw-stage habitats, and seven resulting viromes yielded 53 vOTUs. Viral-specific bioinformatics methods were used to recover viral populations, define their gene content, connect them to other related viruses (globally) and potential hosts (locally). Only 15% of these vOTUs had genetic similarity to publicly available viruses in the RefSeq database, and ∼30% of the genes could be annotated, supporting the concept of soils as reservoirs of substantial undescribed viral genetic diversity. The vOTUs exhibited distinct ecology, with different distributions along the thaw gradient habitats, and a shift from soil-virus-like assemblages in the dry palsas to aquatic-virus-like assemblages in the inundated fen. Seventeen vOTUs were linked to microbial hosts (in silico), implicating viruses in infecting abundant microbial lineages from i Acidobacteria, Verrucomicrobia, and Deltaproteobacteria, including those encoding key biogeochemical functions such as organic matter degradation. Thirty auxiliary metabolic genes (AMGs) were identified and suggested virus-mediated modulation of central carbon metabolism, soil organic matter degradation, polysaccharide binding, and regulation of sporulation. This pilot dataset suggested viral community structure changes with permafrost thaw, and that resident viruses impact ecosystem C processing via killing dominant microbial lineages & modulating several C metabolic pathways. We proceeded to optimize steps for generating quantitatively- amplified viromes for capturing both ssDNA and dsDNA viruses. Three different DNA extraction kits were tested and one yielded appreciable distinct communities (with ~1/3 novel viral populations), suggesting that different DNA extraction kits may bias the viral communities captured. The optimized protocol resulted in increased DNA yield and purity leading to the recovery of 299 vOTUs. Together, these findings suggest that these soil viruses have distinct ecology and impact host-mediated biogeochemistry via top-down (inferred from lysing dominant microbial hosts) and perhaps bottom-up (inferred from virally-encoded metabolic genes) controls. This optimized protocol could now be used to generate quantitatively-amplified viromes and in combination with the initial ecological insights provided here, these data can now guide future research in confirming and predicting viral impacts on soil ecosystem services. ii Dedication Peggy Cianciolo. You were a second mother to me, my first mentor, taught me how to give back, and you pushed me to do better every day. I owe you so much, and will always remember you; R.I.P. Mom. As much as I think I have grown up and push away from you, you are always there when I need you. I could not have completed my Ph.D. without you. I am extremely grateful for you. Brittany. At no time did you ever second-guess my passion for science. Over the years we have metamorphosed into the ultimate team. The problems we have encountered during my graduate career could easily be listed in the Guinness World Records, but we are resilient and have overcome so much. Connor. You defied the odds and brought light into the worst time of our lives. You are a testament that I can overcome my fears and rise to any challenge. When I look at you any doubt or stress I have goes away and I am reminded of my success. You are my everything. iii Acknowledgments I want to thank all collaborating scientists from the IsoGenie Consortium. The majority of this work was supported by the US Department of Energy Office of Biological and Environmental Research (DE-SC0004632 and DE-SC0010580) and the Gordon and Betty Moore Foundation (grant #3790 to MBS). Additionally, the final chapter material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Workforce Development for Teachers and Scientists, Office of Science Graduate Student Research (SCGSR) program. The SCGSR program is administered by the Oak Ridge Institute for Science and Education (ORISE) for the DOE. ORISE is managed by ORAU under contract number DE‐SC0014664. Thanks are extended to Rich and Sullivan lab members for assistance. Thank you to Frank Corsetti, John Spear, Bradley Stevenson, Ann Close, and the rest of the GeoBiology 2014 group for the support and guidance. Thank you to The University of Arizona and the SWES department for some financial support and guidance. Thank you to The Ohio State University and Microbiology department staff, especially Staci Schweinfurth, for support, guidance, and assistance. Thanks to Virginia Rich, you have helped me grow in so many ways, and I am so grateful to have been advised by you. Thanks to Alison Murray for mentoring me, teaching me how to be self-reliant, and how to be a graduate student. Thanks to Peter Cotty for giving me a first great lab experience and pushing me to go to graduate school. A special thanks to Tim Graham and Alice Chi from OSU’s counseling center, being in group counseling gave me an outlet to express myself, learn from others, and remain sane. iv Vita 2011................................................................B.S. Environmental Microbiology, University of Arizona 2013................................................................Outstanding Graduate Student, University of Nevada, Reno 2013................................................................M.S. Environmental Science and Health, University of Nevada, Reno 2014................................................................Sky School fellowship, University of Arizona 2018................................................................DOE Office of Science Graduate Student Research fellowship, Joint Genome Institute 2013 to present ..............................................Graduate Research Associate, Department of Microbiology, The Ohio State University Publications Trubl, G., Jang, H.B., Roux, S., Emerson, J.B., Solonenko, N., Vik, D.R., Solden, L., Ellenbogen, J., Runyon, A.T., Bolduc, B. and Woodcroft, B.J., 2018. Soil viruses are underexplored players in ecosystem carbon processing. mSystems, 3(5), pp.e00076-18. Emerson, J.B., Roux, S., Brum, J.R., Bolduc, B., Woodcroft, B.J., Jang, H.B., Singleton, C.M., Solden, L.M., Naas, A.E., Boyd, J.A. and Hodgkins, S.B., 2018. Host-linked soil viral ecology along a permafrost thaw gradient. Nature microbiology, 3(8), p.870. Trubl, G., Solonenko, N., Chittick, L., Solonenko, S.A., Rich, V.I. and Sullivan, M.B., 2016. Optimization of viral resuspension methods for carbon-rich soils along a permafrost thaw gradient. PeerJ, 4, p.e1999. Brum, J.R., Ignacio-Espinoza, J.C., Kim, E.H., Trubl, G., Jones, R.M., Roux, S., VerBerkmoes, N.C., Rich, V.I. and Sullivan, M.B., 2016. Illuminating structural proteins in viral “dark matter” with metaproteomics. Proceedings of the National Academy of Sciences, 113(9), pp.2436-2441. Fields of Study Major Field: Microbiology v Table of Contents Abstract ........................................................................................................ ………………i Dedication………………………………………………………………………………...iii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii Chapter 1: Introduction ...................................................................................................... 1 Chapter 2: Resuspending viruses from organic-rich soils ................................................ 11 Chapter 3: Characterization of resuspended Stordalen Mire dsDNA viruses ................... 31 Chapter 4: Towards optimized viral metagenomes from challenging soils ...................... 68 Chapter 5: Conclusions ................................................................................................... 111
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