Hyper/thermophiles: 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 biosphere 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 organism 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 halophiles 350°C ¾ pH 3 to 8 in sea water -Mainly neutrophiles ¾ Temperature : 2 to more than 350°C 2°C - Psychrophiles 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 bacteria • Anaerobic and aerobic hyper/thermophiles Small subunit rRNA based universal phylogenetic tree The red bulky lineages represent hyperthermophiles Karl Stetter et al., 2006 Desulfobacterium thermolithotrophicum • Balck smoker, MAR, 3500m • Bacteria • L’Haridon et al., 1998 • motile rod • Chemo-autotroph • e- donnor : H2 • G+C: 35% mol • e- acceptor: S° • 40-70-75°C • pH: 4-6-8 • NaCl: 1.5-3.5-7% • Generation time: 135 min Methanocaldococcus jannaschii • Jones et al., 1983 • Mobile • Black smoker, EPR, 2600 m • G+C: 31% mol • Archaea • T°C: 50-85-86 • Coccoid shape • pH: 5.2-6-7 • Chemolithotrophs • Generation time: 26 min • Methane producer Pyrolobus fumarii • Blöch et al., 1997 • G+C: 53% mol • Black smoker, MAR, 3650 m • 90-106-113°C • Archaea • pH: 4-5.5-6,6 • Coccoid • NaCl: 1-1.7-4% • Chemolithoautotroph • Generation time: 60 min • e- donnor: H2 • Tolerance: 25 MPa, 1h et 2 121°C • e- acceptors: NO3, S2O3 -, O2 Pyrococcus yayanosii Pyrococcus yayanosii sp. nov 1st hyperthermophile obligate piezophile microorganism which has extended the upper limits of life in water on Earth (Pmax 130 MPa and Tmax 110°C) P. furiosus (0m) P horikoshii (1400m) • Ashadze (Serpentine 2007 P. abyssi (2200m) MAR,4500m) the deepest site P. glycovorans (2650m) •Optima 98°C and 52 MPa T barophilus (3550m) P. yayanosii (4100m) Zeng et al, ISME J. 2009 Birrien et al, IJSEM sous presse Souchothèque de Bretagne: collection of Extremophiles 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 biology, 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 Thermus 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 marine life, 18 millions DNA microbial sequences corresponding to 100 phylums (8000 prokaryotic species) are available and it is estimated that the marine microorganisms (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 organisms 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.