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Published February 2018 | Supplemental Material + Accepted Version
Journal Article Open

Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods

Abstract

While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1, and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labeling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.

Additional Information

© 2017 Wiley. Issue online: 14 February 2018; Version of record online: 14 December 2017; Accepted manuscript online: 21 November 2017; Manuscript Accepted: 12 November 2017; Manuscript Revised: 10 November 2017; Manuscript Received: 8 August 2017. We thank K. Dawson and G. Chadwick for many insightful conversations regarding data interpretation and analysis and R. Hatzenpichler and B. Babin for useful discussions regarding BONCAT and click chemistry. We also thank J. Nissomov, F. Natale and J. Latham for their technical assistance with the E. huxleyi-EhV experiments as well as Jennifer Brum and Sarah Schwenck for help exploring the use of TEM microscopy for mapping Syn1 viral particles. This work was funded by the Gordon and Betty Moore Foundation through Grant GBMF3780 to VJO, GBMF3789 to KDB and GBMF3305 and GBMF3790 to MBS as well as NSF Biological Oceanography awards to KT, KDB (OCE-1559179) and MBS (OCE-1536989). ALP was supported by an NSF OCE Postdoctoral Research Fellowship. The Caltech Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through grant GBMF775, the Beckman Institute, and NIH (S10OD020013).

Attached Files

Accepted Version - emi13996.pdf

Supplemental Material - emi13996-sup-0001-Tab-Figs.pdf

Supplemental Material - emi13996-sup-0002-Appendix_20S1.pdf

Supplemental Material - emi13996-sup-0003-Appendix_20S2.pdf

Supplemental Material - emi13996-sup-0004-Appendix_20S3.pdf

Supplemental Material - emi13996-sup-0005-Appendix_20S4.pdf

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Additional details

Created:
August 22, 2023
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