Viruses in the deep subsurfaceBert Engelen

Phages from Rhodobacter capsulatus-affiliated strain E32 ODP Leg 201, Site 1230, sediment depth: 268 mbsf

engelen@icbm.de

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

Hypotheses

Where to drill?

Anywhere !

Contamination controlled, fresh, active & deep samples

Viruses

Image: Häusler, 2007

Virus bacteriophage phage

The most abundant biological entities with 1031 viruses on our planet (Breitbart & Rohwer, 2005)

Up to ~1023 per second viral infections in the oceans (Shuttle, 2007)

Lytic cycle

Induction

Lysogeniccycle

7 hours: no detectable VLP

19 hours: no detectable VLP (Control)

1.2 x 1010 VLP/ml (Mitomycin C)

Control Mitomycin C

10µmSybrGreen I

Induction of prophages fromRhizobium radiobacter strain P007

Control

Induced

Addition ofantibiotic

Incubation

Washing steps

time [h]0 5 10 15 20 25

0

0.5

1

1.5

2

OD

60

0

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)

The viral shunt

Rhizobium radiobacter, isolated from 198 mbsf

Cell

Cell

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

Previous work on benthic phages

Quantification of phages atvarious sampling sites

Continental margin,slope & equat. Pacific

Coastal sediments(Janssand)

Bering Sea

South Pacific

South PacificGyre

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

Virus-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

Continentalmargin

South PacificGyre

Increasing in oligotrophic and deep sedimentsPreservation 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)

Are 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 populationscontrolled by viral infections?

What is the main viral proliferation mode in the terrestrialsubsurface?

What is the viral diversity and the host-specificbiogeography?

How relevant is the viral shunt as a factor for sustainingthe terrestrial deep biosphere?

Can heterotrophic microbial communities thrive on cellcomponents that derive from the viral lysis of autotrophs?

Questions

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 marinedeep biosphere

Work loads