Metagenomic to find and characterize microrganism surviving

in space.

Carlotta MorichiFabian Knöpfel

Frederic GaboyerJudith GendronNuma Lauront

Group 5:

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-.....

-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.

Antartic, Mc Murdo Dry Valley

Space conditions

UV ray

microgravity

space vacuum

Cosmic radiation

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-AUV-BUV-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 vacuum:

Space conditions

microgravity:

« 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

No clear biological problem.

? -nutrition-excretion-motility

Scientific context

• Only few and well known organisms have been investigated :

But model organisms remain minor inregards to the microbial biodiversity.

- Survival of B.subtilis spores :

Unprotected : several secondsProtected : more than 6 years

- Others microorganisms :

Phage T1, SynechococcusHaloarcula, Deinococcus

• Recently : surprising survival of lichen

90-95% microorganisms remain uncultivable in laboratary

1 : Who is here ? Biodiversity caracterization2 : Who does what ? Physiological caracterization

Why using Metagenomics ?2 : Unknown microbial diversity

• BUT with Metagenomics :

Caracterized

Uncaracterized

• With Molecular approaches : 16S/18S rRNA : Biodiversity

Environmental sample all DNA sequencing (Genome informations)

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

Principe of metagenomics

Sample collection

Whole DNA extraction

Whole DNA amplification

Whole DNA sequencing

Data analysis

Information about : Biodiversity but also physiology, metabolic pathways…

Whole DNA extraction

Whole DNA amplification

Whole DNA sequencing

Data analysis(alignement, comparaison

of sequences)

Metagenomics applicated to survival in space

Exposure

Alive cells Dead cells

space conditions

No exposure

Whole DNA extraction

Whole DNA amplification

Whole DNA sequencing

Data analysis

Comparaison

Sample

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

Limitations of metagenomics

MODEL: SYNECHOCOCCUS

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

sample model

Exposed to simulated space conditions

Not exposedcontrol

dead alive

Genessequencing

Genes sequencing

ControlNot exposed

Proteomic

space

Exposed to (simulated) space conditions

Take an other sample froma diffrent place

COMPLEMENTARY APPROCHES

To study adaptations of cells in extrem conditions:

Proteomics study with mass spectromphotometry

Transcriptom

Physiology / Metabolism

Proteom: is the sum of the proteins within a cell at a set point in time under defined parameters .

PROTEOMICS : study of proteom

mRNA

ONE GENEPROTEINS

POST TRANSLATION MODIFICATIONS

SIMULATED SPACE CONDITIONS ON EARTHThe goal of stage 1To specify the expression of every protein under the influence of one parameter

DIRECT EXPOSURE IN SPACEThe goal of stage 2 :To have realitisic conditions and interactions inside the cell (between genes, proteins metabolism)

S1 controle

Sample

COMPARE proteomics

(transcriptomic)

Sample Synechococcus

STAGE 1 : study the SIMULATED conditions on Earth

STAGE 2 : study in the real space environment

Sample

S 2

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

sandstone community

Mac Murdo Dry Valley

Equipments and methods

2 : Exposure to space conditions

- On Earth : - In space :

Aerospace Center, Köln

FOTON Spacecraft (ESA)

BIOPANPlanetary and Space

Simulation facilities (PSI)

3 : Separation of cells : LIVE / DEAD dye kit + FACS

Dead cells : green fluorescenceAlive cells : red fluorescence

2 cellular populations

Equipments and methods

1 : labelling 2 : separation

5 : DNA amplification : Multiple Displacement Amplification (MDA)

Amorces hexamériques, + ADN polymérase du phage Phi29.

Equipments and methods

4 : DNA extraction : Standard and appropriated protocols

6 : DNA sequencing 454 PyrosequencingSequences of 100 – 200 pb

7 : Data analysis : bioinformatic tools :

- BLAST (http://www.ncbi.nih.gov/BLASTsequences from eukaryotes. SILVA aligner (http://www.arb-silva.de, Pruesseet al., 2007)

-KEYDNATOOLS (http://keydnatools.com/)- NCBI databases

Genome construction

Sequence analysis

Phylogenetic Functionnal

Who is here ? Who does what ?= Biodiversity = Physiology

Equipments and methods

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

Extract from: S. Onofri et al. 2008Mimic space conditionsFind the condition that only allow extremelyResistant micro organisme to survive

Experimental conditions and exposure time for gene expression

analysis

Time response to stress exposure

Protein PT regulation

Protein neo synthesis (neo mRNA)

T0 T1 T2 T3 T4T3

Stress conditions

Times of analysis (Ti)

Choose the good time and stress inductions in different conditions

Expected results

Metagenomics in extremes conditions

Proteomics : gene expression studies in extremes conditions

Proteomics approach results

Protein NameT0 Quantities T1 T2 T3 T4

ProtA 0,11 0,12 0,11 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

Proteom or transcriptom

Relative Expressions at T i/T j

proteins/genes probably implicated in stress responses

Graphical representation with 2 conditions

Shape of protein/gene expression results

Protein present only in a stress conditions

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Algorithm

Simplification of the results

Results after simplification of data of a high range analysis in all dimension

Axe 1 (53%)

Axe 2 (24%)

A better understanding of genes implicated in stress responses of

Synechococcus

UV

UV

Vacuum

Temperature

Genes or proteins associated with stress conditions

Chronology of response to the space stress

Now we can investigate physiology and network of these stress responseswith an important data-base.

Post translational modification studies with proteomics data

Expected results : metagenomics

• After analysis BIODIVERSITY

Discovery of : - new species ?- new phyla ?

• Direct results : DNA sequences and contigs

Phylogenetic trees :

Unknown microorganisms Who are they closest to ?

Expected results : metagenomics

Genomes : Genes : Pathways : functional groups metabolism, physiology

After analysis PHYSIOLOGY and GENOMIC

Special attention to :- G+C contents- Genome sizes- DNA repair mechanisms- Pathways of excretion, polysaccharides secretion

……

Applications

Growth Vaccine Secondary Metabolite

Resistance

Protein

Applications

Applications

Danger

Human health Avionics and spacecraft system

Microbs become more pathogenic & resistant to antibiotica

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