Hyper/: biodiversity and potentials

Prof. Mohamed Jebbar

Laboratory of Microbiology of extreme environments University of Brest

The "Rendez-vous de Concarneau", August 30, 2011 The majority of the is under pressure

Prokaryotes : the unseen majority

Total : 4-6 1030 cells, Milieu marin : 1.18 1029 cells euphotic zone : 3.6 1028 cells, deep-sea (>1000m) : 6.5 1028 cells

Only ~8000 prokaryotic species, mainly from terrestrial settings, were described Oger et Jebbar., 2010, Research Microbiol, 161:799, Whitmann et al., 1998, PNAS, 95:6578 Microbial communauties in deep-sea extreme environments

Diversity and functionning

Ashadze

4°S FUTUNA

CIR

Drilling sites , hydrothermal vents, cold seeps and brines, Geothermal sources Access to deep-sea extreme environments:

9 Research Vessels (RV)

9 Submersible and ROV 1977,1979: discovery of deep-sea hydrothermal vents

Riftia pachyptila a fascinating Ashadze sit on MAR 4100 m, two active from deep-sea hydrothermal vents black smokers colonised by anemones

Unexpected biotopes on Earth Hydrothermalism Physico-chemical characteristics of deep-sea hydrothermal vents

¾ Salt concentration (35 g/L) - moderate 350°C

¾ pH 3 to 8 in sea water -Mainly

¾ Temperature : 2 to more than 350°C 2°C - to hyper/thermophiles

¾ Pressure : depends on the deepth - Piezophiles Prokaryotic diversity of deep-sea vents

• Chemo-autotrophs symbionts

• Planctonic Autotrophs and heterotrophs

• Surface associated

• Anaerobic and aerobic hyper/thermophiles Small subunit rRNA based universal phylogenetic tree

The red bulky lineages represent

Karl Stetter et al., 2006 min -7% 3.5 -8 6 -75°C 70 G+C: 35% mol 40- pH: 4- NaCl: 1.5- Generation time: 135 • • • • • , 1998 2

et al.

Desulfobacterium thermolithotrophicum acceptor: S° donnor : H motile rod Chemo-autotroph e- Balck smoker, MAR, 3500m Bacteria L’Haridon e- • • • • • • • -86 -7 85 6 Mobile G+C: 31% mol T°C: 50- pH: 5.2- Generation time: 26 min • • • • • ., 1983

et al

Methanocaldococcus jannaschii Jones Black smoker, EPR, 2600 m Coccoid shape Chemolithotrophs Methane producer • • • • • • -4% -6,6 1.7 -113°C 5.5 106 pH: 4- G+C: 53% mol 90- NaCl: 1- Generation time: 60 min Tolerance: 25 MPa, 1h et 121°C • • • • • • 2 -, O 2 3 O 2 , S 3 2 , 1997

Pyrolobus fumarii et al. acceptors: NO donnor: H Blöch e- Black smoker, MAR, 3650 m Archaea Coccoid Chemolithoautotroph e- • • • • • • • a max

52 MP us presse

ine 2007 t and T and a C °

P , IJSEM so 98

a

ng et al, ISME J. 2009 130 M m

i e t Z Birrien et al p shadze (Serpen max A MAR,4500m) the deepest site piezophile • •O ) (2650m (3550m) (4100m) (1400m) (0m) (2200m) of life in water on Earth (P

obligate P. furiosus P horikoshii P. abyssi P. glycovorans T barophilus P. yayanosii st sp. nov 1 i

nosi

Pyrococcus yayanosii ) C ° 110 Pyrococcus yaya which has extended the upper limits Souchothèque de Bretagne: collection of of LM2E

IFREMER: 1281 isolats including 494 thermophiles and 787 mesophiles.

(677 aerobes, 604 anaerobes, 350 archaea and 930 bacteria).

CNRS/UBO: 485 thermophiles (120 aerobes, 365 anaerobes, 350 archaea and 135 bacteria) and 1435 mesophiles.

Web site: http://www.ifremer.fr/souchotheque/internet/htdocs/generique.php?pagebody =accueil.php Application of hyperthermophiles

Hyper/Thermophiles display original properties that are of high interest to biotechnology.

• Cells in culture can be directly used in some applications like bioleaching or Biofuels.

• More generally, their interest is directly linked to their cellular components like proteins and enzymes, which are used in molecular , detergent, fine chemical, food processing and biofuels industries.

• Biopolymers from extremophiles are also of high interest for different fields including cosmetics.

• Extremolytes produced by Hyper/Thermophiles and other extremophiles are novel products used mainly for macromolecules stabilization.

• Additional perspectives are opened through the use of novel methods like high throughput culturing and synthetic genomics. Venuceane ™

Through biotechnology, Venuceane ™ harnesses the power of thermophilus to bring its benefits to human skin.

VENUCEANE™ a heat stable biotechnological extract containing Extremozymes™ obtained by fermentation.

In use, it offers the dual advantage to be activated by heat and protect against UV, even in vivo in actual weather conditions.

Venuceane is recommended in: Formulations Anti-ageing Formulations during or after tanning. A day care cream and antioxydant treatments Proprerties of Hyper/thermostable DNA polymerases used in PCR DNA polymerases and damaged nucleic acids

9 New molecular tools to amplify damaged DNA

¾ One thermostable DNA polymerase is marketed : DPO4 (famille Y), Trevigen

Amplified Sequences bit reliable Restricted Spectre of amplified damaged DNA Synthesised strands are too short

¾ Amplification of damaged DNA by P. abyssi DNA polymerases

PCR simplex ------

Taq D Dexo B D D Bexo D Dexo Bexo B Dexo B B Dexo Bexo Bexo

75 bp 500 bp 48 bp Mésappariement - - - 0213 3 3’-terminal 0 1 2 0 1 2 3

Possible applications in legacy medicine and paleogenetic… Genome maintenance in hyperthermophilic Archaea Biotechnological aspects

Without RPA RPA 10 µM

0 0 DNase I DNase I 0 RPA

DNA + RPA

ADN

DNA degraded DNA protected Storage 12 mmonths at 12°C

Efficient protection of ssDNA

0 RPA Taq PCR Pfu PCR DNA +-+- +-+- RPA --++ --++

DNA + RPA

DNA

DNA binding activity is conserved After storage 12 months at 12°C Developping a HDA (Helicase –dependent- amplification) technology as a module for DNA Compatible with PCR applications amplification to be used in environmental designed sensors. Large subunit of RPA as a molecular biology reactif. A patent was deposited: amplification of DNA with protection of at ambient T°C Application of hyperthermophiles

Haruyuki Atomi et al., Curr Opi Biotech, 201 Extremolytes applications

Lentzen & Schwartz, 2006 Industries using marine biological ressources

David Leary et al., Marine policy, 2009 Cultivating the uncultured

Less than 1% could be cultured Alain and Querellou 2009 Discovery of new enzymes by High-throughput discovery process (MAMBA project FP7, 2009-2013)

Genomes-metagenomes Protein expression characterization

Enzyme Production

Sampling Sample processing Enrichments Isolates, libraries

Screening platforms Enzyme improvement Culture and isolation automated platform for microbes COCAGNE project (Brittany RC + State) From Census of , 18 millions DNA microbial sequences corresponding to 100 phylums (8000 prokaryotic species) are available and it is estimated that the marine (mainly procaryoetes) of different kinds could represents more than 1 billion. Conclusions • Exploration of life and its properties on Earth is not completed yet

• New biotopes and living forms are still to be discovered

• Physio-chemical and spatial borders of life are not known

• Most of novelties are in the deepest parts of Earth

• Deep sea extremophiles (e.g. hyperthermophiles) is a source of macromolecules with structure and activity adapted to extreme conditions

• Similar biotopes (past or present?) harbouring Prokaryote-like may exist elsewhere within the solar system Acknowledgments

• Laboratory of Microbiology of Extreme Environments UMR 6197 (CNRS, IFREMER, IUEM, UBO) J. Querellou , D. Prieur and all members of the LM2E lab

• School of Life Science & Biotechnology, SJTU, Shanghai, China . X. Xiang, F. Wang, and J. Xu Photo of the Logatchev site at MAR, 3000m depth