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 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 Archaea 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 microorganism • •O ) (2650m (3550m) (4100m) (1400m) (0m) (2200m) of life in water on Earth (P
hyperthermophile 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 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