Metagenomic to find and characterize microrganism surviving in space.

1. Carlotta Morichi Fabian Knöpfel Frederic Gaboyer Judith Gendron Numa Lauront Metagenomic to find and characterize microrganism surviving in space. Group 5:

2. 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 -.....

3. 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.

4. 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

5. 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:

6. 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: -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

7. 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

8. 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.

9. 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

10. Principe of metagenomics Sample collection Whole DNA extraction Whole DNA amplification Whole DNA sequencing Data analysis Information about : Biodiversity but also physiology, metabolic pathways…

11. 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

12. 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

13. MODEL: SYNECHOCOCCUS • Survivals in Space 2 weeks • Sequenced Genome : permit comparaison with data bases • Studiedmuchtime, well known • Resists to dissection

14. 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

15. 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

16. 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)‏

17. 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

18. RESULTS OF MASS SPECTROMETRY: massspectrum: identity card of the protein And even protein sequence

19. Equipments and methods 1 : Sample collection Antarctica environment : dry, cold and submitted to high U.V radiation Mac Murdo Dry Valley sandstone community

20. 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

21. Equipments and methods • 3 : Separation of cells : • LIVE / DEAD dye kit + FACS Dead cells : green fluorescence Alive cells : red fluorescence 2 cellular populations 2 : separation 1 : labelling

22. Equipments and methods 4 : DNA extraction : Standard and appropriatedprotocols 5 : DNA amplification : Multiple Displacement Amplification (MDA) Amorces hexamériques, + ADN polymérase du phage Phi29. 6 : DNA sequencing 454 Pyrosequencing Sequences of 100 – 200 pb

23. 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

24. Mimic space conditions 3 parametres to define : • UV • Vacuum • Temperature The Planetary and space simulation facilities (PSI) in Germany (KÖLN)‏

25. 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

26. 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 T3 T4 Time response to stress exposure Stress conditions

27. Expected results • Metagenomics in extremes conditions • Proteomics : gene expression studies in extremes conditions

28. Protein Name T0 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 Proteomics approach results

29. 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

30. Simplification of the results Axe 2 (24%)‏ Axe 1 (53%)‏ Results after simplification of data of a high range analysis in all dimension

31. Vacuum UV Temperature 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

32. Expected results : metagenomics • Direct results : DNA sequences and contigs • After analysisBIODIVERSITY • Discovery of : • new species ? • new phyla ? Phylogenetic trees : Unknown microorganisms Who are they closest to ?

33. Expected results : metagenomics After analysisPHYSIOLOGY and GENOMIC Genomes :Genes: Pathways : functional groups metabolism, physiology Special attention to : - G+C contents - Genome sizes - DNA repair mechanisms - Pathways of excretion, polysaccharides secretion ……

34. Applications Protein Growth Vaccine Secondary Metabolite Resistance

35. Applications

36. Applications

37. Danger Avionics and spacecraft system Human health Microbs become more pathogenic & resistant to antibiotica Negative impact of immune system Not well understood

38. 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