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
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
Viruses
Virus  bacteriophage  phage
Lytic cycle
The most abundant biological
entities with 1031 viruses on our
planet (Breitbart & Rohwer, 2005)
Lysogenic
cycle
Up to ~1023 per second viral
infections in the oceans (Shuttle, 2007)
Induction
Image: Häusler, 2007
Induction of prophages from
Rhizobium radiobacter strain P007
Control
2
OD600
1.5
Induced
Washing
steps
1
Incubation
0.5
Addition of
antibiotic
0
0
5
10
time [h]
15
20
25
Control
Mitomycin C
7 hours: no detectable VLP
19 hours: no detectable VLP (Control)
1.2 x 1010 VLP/ml (Mitomycin C)
SybrGreen I
10µm
Why are we interested
in phages in the subsurface?
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!
… 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
Cell
Phage capsids
Phage
Cell
Cell
Cell
Production of phage particels
Infection of the host
Rhizobium radiobacter,
isolated from 198 mbsf
100 nm
Free Rhizobiophages
20 µm
DNA released after lysis of the cell
Previous work on benthic phages
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
Quantification of phages at
various sampling sites
Bering Sea
Coastal
sediments
(Janssand)
Continental margin,
slope & equat. Pacific
South Pacific
Gyre
South Pacific
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
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
Are viruses a source of
organic carbon?
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
Questions
To which extent are deep-biosphere
controlled by viral infections?
populations
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?
Work loads
Count viruses, determine
quantify virus production
down
hole
virus-to-cell
ratio,
Analyse viral diversity (metaviromics if enough material available)
Determine presence and diversity of lysogenic phages by phageinduction 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 H2consumers 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
deep biosphere
between
terrestrial,
limnic
and
marine