V iruses in the deep subsurface

1. Viruses in the deep subsurface Bert Engelen Phages from Rhodobacter capsulatus-affiliated strain E32 ODP Leg 201, Site 1230, sediment depth: 268 mbsf engelen@icbm.de

2. Hypotheses • Viral infections appear to control microbial biomass • Viruses supply nutrients to indigenous microorganisms • Organic carbon might be shuttled via viral lysis from H2-consumers to other heterotrophic prokaryotes Where to drill? • Anywhere ! • Contamination controlled, fresh, active & deep samples

3. Viruses Virus  bacteriophage  phage The most abundant biological entities with 1031 viruses on our planet (Breitbart & Rohwer, 2005) Up to ~1023 per secondviral infections in the oceans (Shuttle, 2007) Lytic cycle Lysogenic cycle Induction Image: Häusler, 2007

4. Induction of prophages from Rhizobium radiobacter strain P007 Control 2 1.5 Induced Washing steps OD600 1 Incubation 0.5 Addition of antibiotic 0 0 5 10 15 20 25 time [h] Control Mitomycin C 10µm SybrGreen I • 7 hours: no detectable VLP • 19hours: no detectable VLP (Control) • 1.2 x 1010VLP/ml (Mitomycin C)

5. Phages ... … are mortality factors for prokaryotes: Contribution up to ~71% in the deep ocean (Weinbauer et al., 2003) Up to ~90% at the surface of deep sea sediments (Danavaro et al., 2008) Deep subsurface? Largely unknown! Why are we interested in phages in the subsurface? … exhibit an enormous diversity: ~ 5,000 genomes per seawater sample ~10,000 – 1,000,000 per sediment sample (Edwards & Rohwer, 2005) … provide organic matter via cell lysis: The “viral shunt“ accounts for ~80% of bacterial heterotrophic production in surface sediments (Danavaro et al., 2008)

6. The viral shunt Phage capsids Cell Phage Cell Cell Production of phage particels Cell Rhizobium radiobacter, isolated from 198 mbsf Infection of the host 100 nm Free Rhizobiophages 20 µm DNA released after lysis of the cell

7. Inductionofprophagesfromdeepsubseafloorisolates Half of the tested isolates contained inducible prophages(Engelhardt et al., 2011) Lysogeny might be the main viral proliferation mode in the deep subsurface QuantificationofRhizobiophages in the marine deepsubsurface Rhizobiumradiobacterhighly abundant (~ 5%) (Engelhardt et al., 2013) Site specificdistributionofR. radiobacter(biogeographyofsubpopulations) Rhizobiophages up to 14% of the total virus numbers Previous work on benthic phages

8. Quantification of phages atvarious sampling sites Bering Sea Coastal sediments (Janssand) Continental margin, slope & equat. Pacific South Pacific Gyre South Pacific

9. Virus and cell abundance Virus and cell numbers varied by 4-5 orders of magnitude among different sampling sites Virus and cell abundance decreased with depth

10. Virus-to-cell ratio • Tidal-flat sediments: about 10 (0-5 mbsf) • Continental margin: upto ~20 (>100mbsf) • South Pacific Gyre: upto ~225 (>50mbsf) Tidal-flat South Pacific Gyre Constant in tidal-flat sediments Balance of viral production and decay Increasing in oligotrophic and deep sediments Preservation and ongoing viral production Continental margin

11. 0.2 fg per phage particle (Suttle, 2005) 14 fg per small cell (Kallmeyer et al., 2012) By exceeding a VCR of 70,total biomass consists mainly of viral-bound organic carbon 50% to 80% of the total biomass 20% to 30% of the total biomass Are viruses a source of organic carbon?

12. Questions • To which extent are deep-biosphere populations controlled by viral infections? • What is the main viral proliferation mode in the terrestrial subsurface? • What is the viral diversity and the host-specific biogeography? • How relevant is the viral shunt as a factor for sustaining the terrestrial deep biosphere? • Can heterotrophic microbial communities thrive on cell components that derive from the viral lysis of autotrophs?

13. Work loads • Count viruses, determine down hole virus-to-cell ratio, quantify virus production • Analyse viral diversity (metaviromics if enough material available) • Determine presence and diversity of lysogenic phages by phage- induction experiments (target groups: H2-consuming acetogens, methanogens, sulfate reducers + heterotrophs) • Identify morphologic and phylogenetic diversity of induced phages • Prove viral shunt in growth experiments (cell-lysates of H2- consumers as carbon sources for indigenous heterotrophs) • Test if isolates utilize building blocks of slowly decaying viruses (DNA and proteins as reservoir of bioavailable carbon) • Identify similarities between terrestrial, limnic and marine deep biosphere