Molecular Analysis of the Genetic Diversity and Physiological Status of Natural Picoplanktonic Populations

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Molecular Analysis of the Genetic Diversity and
Physiological Status of Natural Picoplanktonic
Populations
RCC 371

• Oceanic Phytoplankton TeamStation Biologique de
  Roscoff
• UMR 7127 CNRS et Université Paris 6, France

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BIOSOPE Biogeochemistry Optics South Pacific
Experiment
12 November 2004 (Papeete) - 4 January 2005
(Talcahuano, Chili)
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Aims for BIOSOPE1. Mapping the Picoplankton

• Enumerate picophytoplankton cells
  (Prochlorococcus, Synechococcus, picoeukaryotes)
  bacteria /- viruses /-
  photosynthetic anoxygenic bacteria (to be tested)
  by flow cytometry
• gt Volume needed 10 mL all stations / all
  depths
• transect (1 sample / hour)?
• Analyze the cell cycle of Prochlorococcus
  Synechococcus over LD cycles (in combination
  with molecular analyses of stress response)
• gt 4 long-term stations only Volume needed
  10 mL every 3 h for 2-3 days, 7 depths (to be
  discussed)

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Flow Cytometry

• FACSAria
• Laser source 488 635 nm
• High speed cell sorting (gt 5 103 cell./sec)
• Increased Sensitivity
•  Transportable 
• (180 kg!)

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FACSort
ARIA
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Aims for BIOSOPE 2. Genetic diversity

• Isolate new species organisms representative of
  non-cultured environmental groups
• Assess the genetic diversity of (auto- and
  heterotrophic) picoeukaryotes in the euphotic
  hyper-oligotrophic layer (0-200 m)
• Assess the diversity activity of heterotrophic
  picoeukaryotes over the vertical layer (0-2000 m)
• Quantify the abundance of selected taxa and
  identify the dominant species/taxa
• and more (collaborations) Synechococcus,

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Culturing Techniques

• Isolation of new strains (filtration, serial
  dilutions, cell sorting)
• gt Culture Bias might be minimized by cell
  sorting on board
• shortly after sampling
• gt 250 mL per sample (1 station / 3, 4 depths)
• Control of cell presence/growth on board by
• Flow cytometry
• Epifluorescence microscopy
• Characterization (after purification on shore)
• Pigment content (HPLC)
• Morphological (LM TEM)
• Genetic (sequencing 18S rRNA gene)

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Molecular Techniques

• - Pre-screening of genetic diversity of
  picoeukaryotes by TTGE (most stations / 6
  depths)
• gt Volume 5L pre-filtered on 3 µm on-line
  with 0.2µm filter
• - Cloning sequencing 18S 16S rRNA gene from
  natural populations
• (8-10 selected stations depths)
• gt same DNA sample as for TTGE
• - Quantification of the abundance of different
  taxa
• in situ Hybridization (FISH) using 18S 16S rRNA
  probes
• (8-10 selected stations at 6 depths, 1 L)
• Quantitative PCR
• (most stations / 6 depths ?)

- Metagenomics ? (Long-term stations, 2
depths, gt100 L samples, in situ pumps)
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MolecularApproach
in situ Hybridization (FISH)
Natural sample
lt 3 µm
Filtration 0.2 µm Extraction
Cultures
DNA
Amplification 18S/16S rRNA gene
PCR / Quantitative PCR
Clone libraries
DGGE/TTGE
rRNA gene sequencing
Data banks
Phylogenetical analyses
Molecular probes
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Temporal Temperature Gradient Gel Electrophoresis
(TTGE)
Appareillage
Nat. sample
Clone 1
Clone 2
Clone 3
Clone 4
Thermostated bath (60C)
Système Dcode (Biorad)
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An example of DGGE analysis
D. Marie unpubl.
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Example of Clone Library Analysis
Moon et al 2001
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FISH Probes specific of Prasinophyceae
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FISH Abundance of Prasinophyceae off Roscoff
F. Not, unpubl.
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Available Probes for FISH (subset)
Probes in yellow require development
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Quantitative PCR
Natural sample Morlaix Bay
Advantages/FISH faster (96/3 32
samples at a time) more
sensitive (detection lt 0.1 ng d ADN)

Drawbacks/FISH more expensive, do not
visualize cell morphology
measures relative gene abundances, not cell
number
gt 18S rRNA in variable number copies (2-1000 per
cell)

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Q-PCR PROSOPE cruise
Fei Zhu
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Available probes for Q-PCR

• Six primer sets (5 Euk universal, 3Group
  specific)
• EUK Eukaryotes
• CHL Chlorophyta
• MAM (PRAS04) Mamiellales
• MIC Micromonas (not totally specific yet)
• BAT Bathycoccus
• OST Ostreococcus

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Aims for BIOSOPE3. Physiological status of
natural populations

• Expression of specific stress genes (UV stress,
  nutrient starvation) over a LD cycle every 3 h
  for 2-3 days)
• gt at the four long-term stations only, 7
  depths (subsurface,
• 5 m, 10 m, 20m, 30 m, 50 m, DCM
  (Niskin!)
• if bottles 5 L for DNA, 15 L for
  RNA
• if in situ pumps 20L for DNA, 80L
  for RNA
• Characterization of the expression of genes of
  biogeochemical interest in the picoplankton
  (e.g. nifH)
• gt same DNA RNA samples

Problems to be solved with the use of in situ
pumps - Sampling need to be short (only 100 L
necessary) and done as soon as possible after a
CTD cast (within an hour) - flow cytometry
needed will be sampled at same depths with the
rosette (DCM 2 x 10 L) - need for
RNA-free material (use of gloves to handle
filters, RNA-zapping of filter holder,) - need
for pre-tests of RNA extraction from GF/F filters
before the cruise !
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Q-PCR for measuring Gene Expression
Natural sample
Filtration 0.2 µm Extraction
RNA
Reverse transcription
cDNA
Amplification gene X by quantitative PCR
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Analysis of Gene Expression by Q-PCR (Eilat,
Sept. 2000)
Holtzendorff et al. (2001)
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Specific needs
Sample storage - Cruise start (Tahiti)
105-140 L liquid N2 to fill 3 or 4 35-liter
containers Note Samples can be also be
stored at -80C during cruise time but fast
freeze is preferable - Cruise end
(Chili) 50-60 kg dry ice to send all frozen
samples by express mail
in a large cryobox after the
cruise
Pre-cultures will be either brought back  by
hand  in a
cryobox or left on board (requires light and
air
conditioning left on during the transect
back) Space - Beside SBR container lab
space, 1.5 m of bench for filtration ramps Flow
cytometer - Needs to be sent by plane!
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Sample number (estimation)
Euk Syn Hetero. Euks Pro/Syn Pro/Syn
Cyto counts Culture TTGE Clone libraries0-200 m FISH0-200 m Q-PCR Diversity0-200m Clone libraries0-2000 m FISH0-2000 m QPCR genes Cyto Cell cycle
Vol. sampled (mL) 2 250 5 000 1 000 2 000 5 000 1 000 10 000 2
total station 25 12 19 19 19 4 4 4 4
CTD per station 1 1 1 1 1 1 1 8 8
Depth per CTD 12 3 6 6 6 6 12 5 5
Storage container per Depth 1 20 1 4 1 1 2 1 1

Total sample 300 720 114 456 114 24 96 160 160
Storage type N2 or- 80 Live N2 or- 80 -20 or -80 N2 or- 80 N2 ou- 80 -20/-80 N2 or - 80 N2 or -80
Storage containers 2 mL cryotubes Tubes or flasks 2 mL cryotubes filter holders 5mL cryotubes 5mL cryotubes filters 5mL cryotubes 2 mL cryotubes
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Personnel SBR Involved in BIOSOPE

• Daniel VAULOT DR1 CNRS (leg 1)
• Frédéric PARTENSKY DR2 CNRS (leg 2)
• Laurence GARCZAREK CR1 CNRS (leg 1)
• Laure GUILLOU CR2 CNRS
• Nathalie SIMON MdC UPMC
• Dominique MARIE IE CNRS (2 legs)
• Florence Le GALL AI CNRS
• Post-doc X (2003-2004)
• PhD student Y on Diversity (2004-2007) (leg 2)
• DEA or PhD student Z on Physiology (2004-2007)

Collaborations for molecular analyses

• Dave Scanlan, Warwick University diversity
  Synechococcus (DNA/FISH)
• /- John Zehr, USA nifH gene diversity
  (DNA/RNA)
• /- Colomban de Vargas, USA Coccolithophorids
• /- Alex Worden Roseobacter diversity (DNA/RNA)
  ?
• /- Carles Pedros-Alio Archae diversity (DNA) ?
• /- Osvaldo Ulloa, Chili diversity
  eukaryotes, anoxygenic Procs
• /- Z. Kolber, USA anoxygenic bacteria