V iruses in the deep subsurface Bert Engelen Phages from Rhodobacter capsulatus-affiliated strain E32 ODP Leg 201, Site 1230, sediment depth: 268 mbsf

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

V iruses Image: Häusler, 2007 Virus  bacteriophage  phage The most abundant biological entities with viruses on our planet (Breitbart & Rohwer, 2005) Up to ~10 23 per second viral infections in the oceans (Shuttle, 2007) Lytic cycle Induction Lysogenic cycle

7 hours: no detectable VLP 19 hours:no detectable VLP (Control) 1.2 x VLP/ml (Mitomycin C) ControlMitomycin C 10µm SybrGreen I I nduction of prophages from Rhizobium radiobacter strain P007 Control Induced Addition of antibiotic Incubation Washing steps time [h] OD 600

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! W hy 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)

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

Induction of prophages from deep subseafloor isolates Half of the tested isolates contained inducible prophages (Engelhardt et al., 2011) Lysogeny might be the main viral proliferation mode in the deep subsurface Quantification of Rhizobiophages in the marine deep subsurface Rhizobium radiobacter highly abundant (~ 5%) (Engelhardt et al., 2013) Site specific distribution of R. radiobacter (biogeography of subpopulations) Rhizobiophages up to 14% of the total virus numbers P revious work on benthic phages

Q uantification of phages at various sampling sites Continental margin, slope & equat. Pacific Coastal sediments (Janssand) Bering Sea South Pacific Gyre

V irus 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

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

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

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? Q uestions

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: H 2 -consuming acetogens, methanogens, sulfate reducers + heterotrophs) Identify morphologic and phylogenetic diversity of induced phages Prove viral shunt in growth experiments (cell-lysates of H 2 - 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 W ork loads