Metagenomic to find and characterize microrganism surviving in space.

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Metagenomic to find and characterize microrganism
surviving in space.
Group 5

• Carlotta Morichi
• Fabian Knöpfel
• Frederic Gaboyer
• Judith Gendron
• Numa Lauront

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Extreme environments
are known for the hardest parameters that an
organism wich must withstand to survive.Some
exemples are -desert -deep sea
-glacial -halophilic environments -.....
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Antartic, Mc Murdo Dry Valley
-Scientists consider the Dry Valleys perhaps the
closest of any terrestrial environment to Mars,
and thus an important source of insights into
possible extraterrestrial life. -The Dry Valleys
are so named because of their extremely low
humidity and their lack of snow or ice
cove. -Endolithic photosynthetic bacteria have
been found living in the Dry Valleys, sheltered
from the dry air in the relatively moist interior
of rocks.
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Space conditions
Important parameters that we must considerer
. Survival in outer space is reduced due to
damage caused by them to the DNA.
UV ray
Cosmic radiation
space vacuum
microgravity
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Space conditions
Solar UV radiation has been found to be the most
deleterious factor.there are 3 different type of
ray UV-A UV-B UV-C -do not reach the surface
of the earth -in space they are
directly absorbed by the
DNA thymidine containning dimers

cyclobutadipyrimidines
UV ray
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Space conditions
Cosmic rays comes from space, from various
places -Sun -supernova explosions -extremely
distant sources radio galaxies
quasars
Cosmic radiation
Because of their high energy, this type of
particle radiation can be dangerous to people. On
Earth we are mostly shielded from them by our
planet's magnetic field and atmosphere.
One important component of this radiation
comprises the so-colled HZE particles -interactin
g with the atoms of the target causing the
destruction -their high energy they can do a lot
of damage on the subcelluar level -can penetrate
deeper into the body
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Space conditions
space vacuum
 volume of space that is essentially empty of
matter, such that its gaseous pressure is much
less than atmospheric pressure .
-cis-syn cyclobutane
thymine dimers
-trans-syn cyclobutane thymine dimers
Space vacuum UV photoproducts
microgravity
No clear biological problem. ?
-nutrition -excretion -motility
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Scientific context

• Only few and well known organisms have been
  investigated

• Survival of B.subtilis spores
• Unprotected several seconds
• Protected more than 6 years

• Others microorganisms
• Phage T1, Synechococcus
• Haloarcula, Deinococcus

• Recently surprising survival of lichen

But model organisms remain minor in regards to
the microbial biodiversity.
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Why using Metagenomics ?2 Unknown microbial
diversity
Caracterized Uncaracterized

• 90-95 microorganisms remain uncultivable in
  laboratary

Tremendous gap between our knowledge of bacterial
survival in space and microbial abilities

• With Molecular approaches 16S/18S rRNA
  Biodiversity

• BUT with Metagenomics

Environmental sample all DNA sequencing
(Genome informations)?
1 Who is here ? Biodiversity caracterization 2
Who does what ? Physiological caracterization
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Principe of metagenomics
Sample collection
Whole DNA extraction
Whole DNA amplification
Whole DNA sequencing
Data analysis
Information about Biodiversity but also
physiology, metabolic pathways
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Metagenomics applicated to survival in space
Sample
Exposure
No exposure
space conditions
Alive cells
Dead cells
Whole DNA extraction
Whole DNA extraction
Whole DNA amplification
Whole DNA amplification
Comparaison
Whole DNA sequencing
Whole DNA sequencing
Data analysis (alignement, comparaison of
sequences)?
Data analysis
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Limitations of metagenomics
But metagenomics is a global analysis ? many
data How to associate the presence of a gene
with the ability to survive ?
- We need more precise informations - A model
organism is welcome
Synechococcus
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MODEL SYNECHOCOCCUS

• Survivals in Space 2 weeks
  
• Sequenced Genome permit comparaison with data
  bases
  
  
• Studied much time, well known
  
• Resists to dissection

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sample
model
Take an other sample froma diffrent place
Exposed to (simulated) space conditions
Not exposed control
Exposed to simulated space conditions
Control Not exposed
dead alive
Genes sequencing
Proteomic
Genes sequencing
COMPLEMENTARY APPROCHES
space
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To study adaptations of cells in extrem
conditions

• Proteomics study with mass spectromphotometry
• Transcriptom
• Physiology / Metabolism

PROTEOMICS study of proteom
Proteom is the sum of the proteins within a
cell at a set point in time under defined
parameters .
mRNA
ONE GENE
PROTEINS

POST TRANSLATION MODIFICATIONS
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Sample Synechococcus
STAGE 1 study the SIMULATED conditions on
Earth STAGE 2 study in the real space
environment
Sample
Sample
S1 controle
S 2
SIMULATED SPACE CONDITIONS ON EARTH The goal of
stage 1 To specify the expression of every
protein under the influence of one parameter
DIRECT EXPOSURE IN SPACE The goal of stage 2
To have realitisic conditions and interactions
inside the cell (between genes, proteins
metabolism)?
COMPARE proteomics (transcriptomic)?
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WHY MASS SPECTROMETRY?

• sample of isolated specie (i.e syneccococus).
• Caraterise
• the identity of proteins at a set point in time
• This can be repeated at
• Different times
• And under various conditions.

CHROMOTOGRAPHY
MASS SPECTROMETRY
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RESULTS OF MASS SPECTROMETRY mass spectrum
identity card of the protein
And even protein sequence
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Equipments and methods
1 Sample collection Antarctica environment
dry, cold and submitted to high U.V radiation
Mac Murdo Dry Valley
sandstone community
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Equipments and methods

• 2 Exposure to space conditions
• On Earth - In space

Planetary and Space Simulation facilities (PSI)?
BIOPAN
FOTON Spacecraft (ESA)?
Aerospace Center, Köln
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Equipments and methods

• 3 Separation of cells
• LIVE / DEAD dye kit FACS

Dead cells green fluorescence Alive cells red
fluorescence
2 cellular populations
1 labelling
2 separation
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Equipments and methods
4 DNA extraction Standard and appropriated
protocols
5 DNA amplification Multiple Displacement
Amplification (MDA)
Amorces hexamériques, ADN polymérase du phage
Phi29.
6 DNA sequencing 454 Pyrosequencing Sequences
of 100 200 pb
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Equipments and methods

• 7 Data analysis
• bioinformatic tools
• - BLAST (http//www.ncbi.nih.gov/BLAST
• sequences from eukaryotes.
• SILVA aligner (http//www.arb-silva.de, Pruesse
• et al., 2007)?
• KEYDNATOOLS (http//keydnatools.com/)?
• NCBI databases

Genome construction
Sequence analysis
Phylogenetic Functionnal Who is here ? Who
does what ? Biodiversity Physiology
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Mimic space conditions

• 3 parametres to define
• UV
• Vacuum
• Temperature

The Planetary and space simulation facilities
(PSI) in Germany (KÖLN)?
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Conditions for Metagenomics selection
The example of pre ISS exposure Test on black
fungi
Mimic space conditions Find the condition that
only allow extremely Resistant micro organisme to
survive
Extract from S. Onofri et al. 2008
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Experimental conditions and exposure time for
gene expression analysis
Choose the good time and stress inductions in
different conditions
Protein PT regulation
Protein neo synthesis (neo mRNA)?
Times of analysis (Ti)?
T0
T1
T2
T3
T4
T3
Time response to stress exposure
Stress conditions
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Expected results

• Metagenomics in extremes conditions
• Proteomics gene expression studies in extremes
  conditions

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Proteomics approach results
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Shape of protein/gene expression results
Protein present only in a stress conditions
Relative Expressions at T i/T j
proteins/genes probably implicated in stress
responses
. . .
. . .
Proteom or transcriptom
Graphical representation with 2 conditions
Algorithm
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Simplification of the results
Axe 2 (24)?
Axe 1 (53)?
Results after simplification of data of a high
range analysis in all dimension
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A better understanding of genes implicated in
stress responses of Synechococcus
Genes or proteins associated with stress
conditions
UV
Chronology of response to the space stress
Now we can investigate physiology and network of
these stress responses with an important
data-base.
Post translational modification studies with
proteomics data
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Expected results metagenomics

• Direct results DNA sequences and contigs

• After analysis BIODIVERSITY

• Discovery of
• new species ?
• new phyla ?

Phylogenetic trees Unknown microorganisms
Who are they closest to ?
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Expected results metagenomics
After analysis PHYSIOLOGY and GENOMIC
Genomes
Genes Pathways

functional groups metabolism,
physiology
Special attention to - GC contents - Genome
sizes - DNA repair mechanisms - Pathways of
excretion, polysaccharides secretion

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Applications
Protein
Growth
Vaccine
Secondary Metabolite
Resistance
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Applications
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Applications
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Danger
Avionics and spacecraft system
Human health
Microbs become more pathogenic resistant to
antibiotica
Negative impact of immune system
Not well understood
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Conclusion

• A strong expirmental project which
• Has a huge potential of applications
• Could answer more fundamental biological
  questions such as
• - Microorganisms physiology and diversity
  
• - Lithopanspermia therory