Transcript Metagenomic to find and characterize microrganism

Metagenomic to find and
characterize microrganism surviving
in space.
Group 5:
Carlotta Morichi
Fabian Knöpfel
Frederic Gaboyer
Judith Gendron
Numa Lauront
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
-.....
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.
Space conditions
UV ray
Cosmic radiation
space vacuum
microgravity
Important parameters that
we must considerer .
Survival in outer space is
reduced due to damage
caused by them to the
DNA.
Space conditions
UV ray:
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
Space conditions
Cosmic radiation:
Cosmic rays comes from space, from various places:
-Sun
-supernova explosions
-extremely distant sources
radio galaxies
quasars
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:
-interacting 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
Space conditions
space vacuum:
« volume of space that is essentially empty of matter, such
that its gaseous pressure is much less than atmospheric
pressure ».
Space vacuum + UV = photoproducts
-cis-syn cyclobutane thymine dimers
-trans-syn cyclobutane thymine dimers
microgravity:
No clear biological problem.
?
-nutrition
-excretion
-motility
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.
Why using Metagenomics ?
2 : Unknown microbial diversity
Caracterized

90-95% microorganisms remain uncultivable in laboratary
Uncaracterized
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
1 : Who is here ?
2 : Who does what ?
all DNA sequencing
(Genome informations)
Biodiversity caracterization
Physiological caracterization
Principe of metagenomics
Sample collection
Whole DNA extraction
Whole DNA amplification
Whole DNA sequencing
Data analysis
Information about : Biodiversity but also physiology, metabolic pathways…
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
Data analysis
(alignement, comparaison
of sequences)
Whole DNA sequencing
Data analysis
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
MODEL:
SYNECHOCOCCUS
Survivals in Space 2 weeks
 Sequenced Genome : permit comparaison with
data bases
 Studied much time, well known
 Resists to dissection

sample
model
Take an other
sample froma diffrent
place
Exposed to
simulated space conditions
dead
Not exposed
control
Exposed to
(simulated) space conditions Control
Not exposed
alive
Genes
sequencing
Genes
sequencing
space
Proteomic
COMPLEMENTARY
APPROCHES

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
Sample Synechococcus
: study the
SIMULATED
conditions on Earth
STAGE 1
Sample
: study in the
real space
environment
Sample
STAGE 2
S2
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)
S1 controle
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
RESULTS OF MASS SPECTROMETRY:
mass spectrum: identity card of the protein
And even
protein
sequence
Equipments and methods
1 : Sample collection
Antarctica environment : dry, cold and submitted to high U.V radiation
Mac Murdo Dry Valley
sandstone
community
Equipments and methods
2 : Exposure to space conditions
- On Earth :
Planetary and Space
Simulation facilities (PSI)
-
In space :
BIOPAN
FOTON Spacecraft
(ESA)
Aerospace Center, Köln
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
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
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)
Genome construction
-KEYDNATOOLS (http://keydnatools.com/)
- NCBI databases
Sequence analysis
Phylogenetic
Functionnal
Who is here ?
= Biodiversity
Who does what ?
= Physiology
Mimic space conditions
3 parametres to define :
 UV
 Vacuum
 Temperature
The Planetary and space simulation facilities (PSI) in
Germany (KÖLN)
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
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
Stress
conditions
T1
T2
T3
T4
Time response to
stress exposure
Expected results


Metagenomics in extremes conditions
Proteomics : gene expression studies in
extremes conditions
Proteomics approach results
T0
Protein Name
Quantities
ProtA
T1
T2 T3
0,11 0,12 0,11
T4
0,11 0,11
ProtB
1,2
1
1
1
1
ProtC
2
2
2
2
2,1
ProtD
0
5
3
3,2
3,6
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
Simplification of the results
Axe 2 (24%)
Axe 1 (53%)
Results after simplification of data of a high range analysis in all
dimension
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 database.
Post translational modification
studies with proteomics data
Vacuum
UV
Temperature
Expected results : metagenomics
• Direct results : DNA sequences and contigs
• After analysis
Phylogenetic trees :
Unknown microorganisms
Who are they closest to ?
BIODIVERSITY
Discovery of :
- new species ?
- new phyla ?
Expected results : metagenomics
After analysis
Genomes :
PHYSIOLOGY and GENOMIC
Genes :
functional groups
Special attention to :
- G+C contents
- Genome sizes
- DNA repair mechanisms
- Pathways of excretion, polysaccharides secretion
……
Pathways :
metabolism, physiology
Applications
Protein
Growth
Vaccine
Secondary
Metabolite
Resistance
Applications
Applications
Danger
Human health
Microbs become more
pathogenic & resistant
to antibiotica
Avionics and
spacecraft system
Negative impact of
immune system
Not well
understood
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