Download A) Why bacterial diversity - CMIMA

Bacterial single-cell
approaches to the
relationship between
diversity and function
in the Sea
EVK3-CT2002-00078
November 2002 - October 2005
Marine Biodiversity Cluster Meeting - Brussels-July’03
Image: K. Jürgens
1 µm
The project’s ultimate goal
Roundicoccus
southamptii
Dalibacter
banyuleus
75% of BCD
preferentially
grazed by HNF
very sensitive
to viral attack
dominates
DMSP
uptake
Spirovibrio
kalmariensis
Tinymonas
bremenensis
(all names are fiction)
Bacterial single-cell
approaches to the
relationship between
diversity and function
in the Sea
B) Bacterial diversity
A) Bacterial biogeochemical
function
linkage
C) Single-cell approaches
By developing new methodologies, sampling different
European seas and through laboratory and mesocosm
experiments, we will address the main objectives of
BASICS:
The identification of the most important prokaryotic
organisms associated with the biogeochemical
functioning (in the carbon and sulfur cycling) of the
sea, through the development of single-cell analysis
techniques applied to pelagic microbes. BASICS will
also study how resilient the link is between the
diversity and the C and S biogeochemical cycling by
bacterioplankton, in the face of the most important
global environmental changes expected in European
coastal waters.
Objective 1: To describe bacterioplankton diversity in the
coastal seas of Europe
Objective 2: To describe the seasonal changes in the cycling
of carbon and sulfur mediated by planktonic bacteria in
surface waters of European coastal seas
Objective 3: To design, test and fine-tune different methods
and research strategies for the single-cell analysis of
natural bacterioplankton organisms
Objective 4: To link bacterial diversity and biogeochemical
function (in the cycles of C and S) and identify the bacterial
phylotypes responsible for the crucial steps in oceanic
biogeochemical cycling, and to refine recently developed
conceptual frameworks for the links between species
richness (number of dominant coexisting species) and
biogeochemical cycling
Objective 5: To estimate the effect of environmental
changes affecting the ocean’s bacterially-mediated
biogeochemical function, global bacterial diversity and the
link between bacterial diversity and C and S cycling
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
Bacteria are...
•
•
•
•
•
Most of Earth’s living biomass
Most abundant living particles in the sea
Only significant DOM transformers
Responsible for most of ocean’s respiration
Largest living surface in the ocean
• The largest “unknown pool” of genomic and
metabolic (i.e. functional) diversity
• bacteria play far more important ecological roles in natural
environments than their small sizes would suggest (Brock et al.’88)
• “L’essentiel est invisible pour les yeux” (Antoine de Saint-Exupéry)
• “small is beautiful !”
Chisholm 2000
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
Trying to make VISIBLE what
is invisible
Bacteria
Archaea
Stéphan Daigle
National Geographic
Hey, it’s me !
Pure culture
Genes
Proteins
Activity
Molecular
biology
Culturing native prokaryotes
SYSTEM
CULTURABILITY (%)
Marine
0.001 - 0.1
Freshwater
0.25
Mesotrophic lake
0.1 - 1
Estuary
0.1 - 3
Activated sludge
1 - 15
Sediments
0.25
Soil
0.3
Are these few isolated bacteria relevant in plankton biogeochemistry ?
Overview of techniques in molecular ecology
Sea water microbes
In situ
hybridization
Detection
single cells
Biomass collection
Microbial biomass
Nucleic acid extraction
Comparison
among
communities
DNA reassociation kinetics
DNA cross-hybridization
Fingerprinting: lmw RNA
Bulk nucleic acid extract
Quantitative rRNA
hybridization
Abundance
some
phylotypes
PCR amplification
Comparison
among
communities
Fingerprinting
DGGE
ARDRA
T-RFLP
PCR product
Cloning and sequencing
Species
composition
BASICS partners will follow the seasonality
of bacterial diversity in several sites in the
North, Mediterranean and Baltic Seas and
the English Channel
We will use a variety of techniques:
- fingerprinting techniques (DGGE, T-RFLP, SSCP...)
- detection of single phylotypes/groups (FISH)
- cloning and sequencing
- culture isolation
Objective 1: To describe bacterioplankton diversity in
the coastal seas of Europe
• Describe seasonality in “diversity” (what is there, who’s
the most abundant)
• Usage of different techniques (fingerprinting &clon
libraries & isolation...)
• Common framework
• Characterization of isolates
• Biotechnological exploitation of the isolates
The BactLib
(Bacterial
Culture database)
The ecologically-referenced
phylotype database
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
To know the main
routes of C circulation
is a prerequisite...
JGOFS
... for understanding
the fluxes of C in the ocean
It is the belief of BASICS that too much
effort has been put in the past in describing
bacterial diversity in the ocean...
... barely telling what the position and
depth of the sample was...
How can we understand the role that specific
bacteria will play in nature then ???
Fig. 2.2. The feedback linking oceanic
plankton and climate through the
production of atmospheric sulfur. The
original hypothesis postulated that
production of dimethylsulfide (DMS)
by phytoplankton, and its subsequent
ventilation and oxidation in the
atmosphere feeds cloud condensation
nuclei in marine stratus, thereby
increasing cloud albedo. If the
consequent reduction in solar
irradiance forced phytoplankton
toproduce less DMS, then a negative
feedback would operate, thus
stabilizing climate. Recent advances
suggest that it is not only
phytoplankton but the whole food web
(with bacteria playing a crucial role)
that releases DMS.
Kiene et al. 2000
- DMSP is a labile organic molecule which can represent 15% of BCD
and close to 100% of S demand
- DMS participates in climate feedback
BASICS partners will follow the seasonality
of microbial biogeochemical cycling in several
sites in the North, Mediterranean and Baltic
Seas and the English Channel
We will measure a large amount
of stocks and rates:
- Bulk DOC and nutrients
- Algal activity and biomass
- Viral and protozoan stocks and activity
- DMS and DMSP stocks and rates
- etc.
Objective 2: To describe the seasonal changes in the cycling
of carbon and sulfur mediated by planktonic bacteria in
surface waters of European coastal seas
• Seasonal studies in C and S cycling
• Key biogeochemical steps little studied
Ocean Projects in IGBP II
BASICS
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
P-Z-N Dynamics: Populations Change through Time
T. Michaels
Nitrate
Zoop
Phyto
Zooplankton
Phytoplankton
Phyto
T. Michaels
Does it matter what biology is hidden within each box?
Diatoms
Prasinophytes
Prymnesiophytes
Prochlorococcus
Synechococcus
Phyto
Zoo
Phyto
Dynamic Green Ocean Model
Fe
Si
PO
NO3
NH4
4
coccolith.
N2 fixers
diatoms
CaCO3
DMS
producers
Nano
phytoplankton
DOM
Buitenhuis et al. 2003
Biogeochemical fluxes are a function of community structure
MicroZoo
T. Michaels
PicoPhyto
NanoPhyto
Bacteria
Bacteria & &
Nutrients
Nutrients
Stays Suspended
MesoSALPS
Zoo
MicroPhyto
Sinking
Sinking
Particles
Particles
Fecal Pellets
salp
D. Steinberg
copepod
euphausiid
1 mm
Bacteria are abundant and important
But
We are unable of grouping them in
biogeochemically relevant and distinct
“boxes”
Because
we don’t know whether they all do the same, or not...
Objective 4: To link bacterial diversity and
biogeochemical function (in the cycles of C and S) and
identify the bacterial phylotypes responsible for the
crucial steps in oceanic biogeochemical cycling, and to
refine recently developed conceptual frameworks for
the links between species richness (number of
dominant coexisting species) and biogeochemical
cycling
• Multistep strategy
• Coocurrence analysis (Synthesis workshop !)
• Design of oligonucleotide probes
• Test of BGQ function in isolates
• Test during an experimental algal bloom
• Each approach is partial and has some risk of failure
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
Objective 3: To design, test and fine-tune different
methods and research strategies for the single-cell
analysis of natural bacterioplankton organisms
• Flow cytometry sorting: standarization & controls
• FISH improvements
• Combination of techniques: MicroFISH, MicroACT, etc...
• Capillary electrophoresis
• X-Ray microanalysis
• .... ???
The power is in the combination of methods
Fluorescence in situ Hybridization:
("phylogenetic staining")
Environmental sample
Extracted nucleic acids
DNA
rRNA
Hybridized
Nucleic acid probe
rDNA clones
rDNA
Sequences
Comparative Analysis
rDNA
database
Hybridization
MPIMM
Sequencing
DAPI-stained
MicroFISH
35S
DMSP
Roseobacter + AU
ICM
DAPI + AU
Aminoacids
Protein
% active cells
60
C
40
g
20
0
60
C
a
g
a
20
g
20
a
0
a
g
40
40
60
0
Atlantic
Ocean
CC
0
20
40
60
% cells in sample
HMW-DOM
Cytophaga
g-Proteobacteria
LMW-DOM
a-Proteobacteria
Cottrell & Kirchman 2000
Cell sorting by FCM
Prelabeling
Radioactive substrates
Nucleic acid probes
Physiological probes
Laser
(488 nm)
Further analyses of sorted fractions
Trash
FACSCalibur
OOB
FACSVantage
High speed cell sorter
* Activity (radioactivity)
* Identification
* Isolation
* Chemical analyses (C; N; P,….)
PML/SOC/MPIMM
DMSP producing phytoplankton bloom in the North Sea
Emiliania huxleyi y Prorocentrum minimum
Flow Citometry
Blue fluorescence (DNA)
103
FISH
Cytophaga/Flavovacterium
Roseobacter
102
SAR86
101
100
Threshold
101
102
103
Red fluorescence (protein)
Abundance highly
correlated with DMSP
consumption
Zubkov et al. 2001
PML/SOC/MPIMM
Capillary electrophoresis
Basic scheme
Capillary
Absorbance detector
Data recording
Sample
+
Power Supply
Inlet buffer reservoir
NIOZ
Outlet buffer reservoir
Protein separation
+
+
+
+
+
++
+
SDS
-
-
+
- -
- -
Detection
window
-
+
Sample
-
+
Injection
-
+
Separation
-
+
Detection
NIOZ
UV
absorbance
Objective 5: To estimate the effect of
environmental changes affecting the ocean’s
bacterially-mediated biogeochemical function,
global bacterial diversity and the link between
bacterial diversity and C and S cycling
• Functional redundancy, ecosystem stability...
... effects of env. change on
- diversity
- BGQ function
- their linkage
• Environmental perturbations in microcosms
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
WS0: Coordination
WP1: Seasonal
studies of
bacterial
diversity
WP2: Explotaition of
phylotypes and isolate
information
WP3: Seasonal
studies of
biogeochemical
C & S cycling
WP 4: SCA
method
development
Phase 1
WS1: Synthesis WS on bacterial
diversity in coastal euro pean seas
and C & S cycling
Phase 2
WP5: Linking bacterial diversity
with biogeochemical function
WP6: Experimental determination of
the factors that regulate the link
between bacterial diversity and
function
WS2: Mesocosm
experiment
WS3: Synthesis
WS on SCA
WP7: Functional stability of the link
facing global change
WS4: Microcosm
experiments
WS5: Summary
Phase 3
WP1:
Diversity
WP2:
Exploitation
WP3:
C/S-Cycling
WP4: SCA dev
WP5: Linking
WP6:
Factors regulate
WP7: Funct. stabil.
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
Bergen
Kalmar
Plymouth
Southampton
Bremen
Texel
Toulouse
Villefranche
Banyuls
Barcelona
Barcelona
Pep Gasol / Rafel Simó
Banyuls
Philippe Lebaron
Texel
Gerhard Herndl
Kalmar
Åke Hagström
Toulouse
Pascal Bordat
Villefranche
Markus Weinbauer
Plymouth
Steve Archer
Bremen
J. Pernthaler / B. Fuchs
Bergen
Frede Thingstad / Mikal Heldal
Southampton
Mike Zubkov / Peter Burkill
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
• Shelf = 20% of Ocean NPP; supports 90% of Marine
Fisheries Production
• Coastal population = 2.2 billion (40% of total)
Before fishing
• Nutrient inputs
After fishing
(Jackson et al. 2001)
microbialization of food chains
Sampling sites
• Blanes Bay (ICM)
• Banyuls - MOLA & MILA station (OOB)
• Baltic proper landsort (UNIK)
• North Sea Texel site (NIOZ)
• Villefranche point “B” (LOV)
• L4 English Channel station (PML & SOC)
• Helgoland site G (MPIMM)
General characteristics of Blanes Bay
• Typical Mediterranean waters: warm, salty and nutrient-poor
• Oligotrophic coastal system (annual average chlorophyll of 0.5 µg l-1)
• Relatively unaffected by human or freshwater influence
• Separated from oceanic waters by a southwest current associated with a
front in the continental slope (10-20 miles offshore)
• Episodic intrusions of oceanic waters caused by the Blanes canyon
Half mile from
harbour
Depth of 20 m
ICM
Seasonality of
phytoplankton
Main peak of chlorophyll a
during late winter, driven
by high atmospheric
pressures together with
irradiances and
temperatures higher than
similar latitudes in the
Atlantic
ICM
Seasonal succession of bacterioplankton
3 September
2July
29 July
9 October
Summer
Fall
4 November
1 Desember
26 March
29 April
Spring
3 June
27 January
22 Desember 97
25 February
5 February
12 February
19 February
ICM
3 March
1.0
18 March
11 March
Winter
Temporal dynamics of bacterial
groups and populations
ICM
How representative is the Blanes site?
CO
Blanes-Jul
Blanes-Nov
Barcelona-Jul
Masnou-Jul
Masnou-Nov
Harbour-Jan
Barcelona-Jan
Masnou-Jan
Barcelona-Nov
CC
Harbour-Apr
Barcelona-Apr
Masnou-Apr
Four sites 70 km appart
Blanes-Apr
One sample for season
Blanes-Jan
Comparison of bacterial composition
by
Harbour-Jul
Harbour-Nov
DGGE
ICM
1.0
Summer
Winter
Spring
PML/SOC seasonal sampling
site
Time series station since 1988
(Roger Harris and PML
Zooplankton group)
Measurements: (weekly
throughout the year)
•Environmental: Chl a, Temp,
Salinity, Optics, POC, PON
•Biological: Phytoplankton,
Zooplankton, Bacteria, Viruses
•Processes: Primary production
(FRRF), Calanus egg production,
L4 location:
PML/SOC
Western English
Channel
-) Why bacteria ?
A) Why bacterial diversity ?
B) Why biogeochemical function ?
• why the C cycle
• why the S cycle
C) Why study the linkage ?
D) The key: Single-Cell methods
E) Project organization
F) Project partnership
• The sampling sites
• A few of the first results
MPIMM
Horseradish-peroxidase-labeled FISH probes
and catalyzed reporter deposition (CARD)
(tyramide signal amplification, TSA)
fluorescently
labeled tyramide
HRP
HRP
HRP
HRP
protein
Permeabilization
MPIMM
Hybridization Signal Amplification
conventional FISH
FISH & signal amplification
10 seconds
1 second
Sediment
10 seconds
1 second
Surface
water
MPIMM
Exposure time:
Euryarchaeota in coastal North Sea surface waters
DAPI-staining
G2 Euryarchaea
Coastal North Sea,
% detection by FISH
Probe detection rate
[% of total cells]
50
G2-Euryarchaeota
40
30
20
10
0
MPIMM
03
05
07
Month
09
11
01
And still, other unplanned BASICS returns...
• Development and optimization of molecular tools (useful
with other microorganisms and in other ecosystems
• Better knowledge of the spatial and temporal scales of
bacterial biodiversity changes
• Applicability of the “key species” concept to bacteria
• Autoecology of bacteria
• Indicator bacterial species of ecosystem perturbations
• Biotechnological exploitation of effort made at the “pure
science” (knowledge for its own sake) level
• ...
Bacterial single-cell
approaches to the
relationship between
diversity and function
in the Sea
Bacterial diversity
Bacterial biogeochemical
function
linkage
Single-cell approaches
www.icm.csic.es/bio/projects/basics