BIODEEP MEETING - Marseille, April 2002 Workpackages 4 and 5 - Fabienne MOREL, Sam DUKAN Objectives : isolation of new strains from DHABs Interface Brine - brine - brine g/l yeast extract - brine diluted 1/3 - brine diluted 1/ g/l yeast extract - Postgate derived medium (100 g/l NaCl) - ZoBell derived medium (100 g/l NaCl) anaerobiosis 13 °C atmospheric pressure / 360 bars 3/ isolation of microbes Methodology 1/ inoculation of samples 2/ enrichment cultures on agar plates : brine diluted 1/3 supplemented with 5 g/l yeast extract or Postgate or ZoBell derived medium aerobiosis / anaerobiosis room temperature

Results16 isolates : - 7 from BANNOCK - 5 from DICOVERY - 4 from URANIA - 0 from L’ATALANTE BIODEEP MEETING - Marseille, April 2002 Workpackages 4 and 5 - Fabienne MOREL, Sam DUKAN

11 isolates from interfaces : 6 from BANNOCK, 4 from URANIA, 1 from DISCOVERY 2 isolates from brines : 1 from DISCOVERY, 1 from BANNOCK 3 isolates from DISCOVERY sediment BIODEEP MEETING - Marseille, April 2002 Workpackages 4 and 5 - Fabienne MOREL, Sam DUKAN

2 isolates obtained with BANNOCK brine as enrichment culture medium 8 isolates obtained with diluted brine : 5 from BANNOCK, 3 from URANIA 6 isolates obtained with chemically defined media : 5 from DISCOVERY, 1 from URANIA BIODEEP MEETING - Marseille, April 2002 Workpackages 4 and 5 - Fabienne MOREL, Sam DUKAN

2 isolates obtained after enrichment culture at 360 bars : from BANNOCK interface BIODEEP MEETING - Marseille, April 2002 Workpackages 4 and 5 - Fabienne MOREL, Sam DUKAN

11 isolates obtained in aerobiosis : 8 from interfaces (4 from BANNOCK, 3 from URANIA, 1 from DISCOVERY) 2 from brines (DISCOVERY and BANNOCK) 1 from DISCOVERY sediment 5 isolates obtained in anaerobiosis : 3 from interfaces (2 from BANNOCK, 1 from URANIA) 2 from DISCOVERY sediment BIODEEP MEETING - Marseille, April 2002 Workpackages 4 and 5 - Fabienne MOREL, Sam DUKAN

Isolation by successive streaking on agar plates - in aerobiosis - under GasPak-generated anaerobic atmosphere 20 strains Molecular characterization : PCR amplification of 16S rDNA - eubacteria - pure strains Preliminary physiological characterization : - facultative anaerobes - growth on brine / Postgate / ZoBell culture media exception of 2 strains isolated on Postgate derived medium and unable to grow on brine medium 1 strain isolated on brine medium and unable to grow on ZoBell derived medium - growth on Postgate medium containing 50 g/l NaCl : moderate halophiles / halotolerant BIODEEP MEETING - Marseille, April 2002 Workpackages 4 and 5 - Fabienne MOREL, Sam DUKAN

Discussion and Conclusion Majority of isolates obtained - from interface - in media made with diluted brine - in aerobiosis - moderate halophiles / halotolerant - aerobes / facultative anaerobes 2 isolates obtained after enrichment under high pressure conditions  barophiles / barotolerant 5 isolates obtained in anaerobiosis  strict anaerobes Characterization of the 20 isolated strains : - molecular identification by sequencing of 16S rDNA - physiological characterization : growth ability in different conditions of temperature, salinity, pressure utilization of different carbon sources BIODEEP MEETING - Marseille, April 2002 Workpackages 4 and 5 - Fabienne MOREL, Sam DUKAN

Additional Work (Workpackage 7)Screening of total bulk DNA for direct retrieval of genes of interest Concentration of samples by filtration on 0.2  m membranes  DNA extraction Construction of DNA libraries in E. coli - in plasmid vectors  unsatisfying (number of recombinant clones / size of DNA inserts) - in a cosmid vector : insertion of large DNA fragments (on course)