CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare Partner 1c DISTAM Università degli Studi di Milano Daniele Daffonchio Tullio Brusa Sara Borin Adriana Bona Enrica Canzi Claudia Sorlini WP6: genetic characterization of isolated microorganisms and of enzymes interesting for biotechnological application

Catabolic genes characterization 19% of the isolates are able to grow on oil and catechol as unique carbon sources Research for xyl E-type genes (catechol 2,3 dioxigenase) gene via PCR Isolated strains Community DNA All negatives DHABs cultivable/uncultivable bacteria do not clevage aromatic ring via common extradiol cleavage

Plasmid profile characterization Plasmid profile has been characterised for several 2001 aerobic isolates Plasmids (mobile genetic elements?) are abundant among Discovery isolates, and absent in L’Atalante strains Plasmids are equally distributed between the most represented phylogenetic groups

“Fishing” of functional genes in the metagenomic DNA Plasmids exogenous isolation Plasmids donor: cells recovered filtrating Discovery water/brines interface concentrated Plasmids hosts: Pseudomonas putida Search for transconiugants donor host MATING Screening for: Hg resistance NaCl tolerance Oil degradation Naphtalene degradation NEGATIVE

Screening for natural competent strains Horizontal gene transfer (HGT) occurs in many natural environments Natural competent cells are naturally able to acquire naked DNA via natural transformation Extreme conditions imposed by the brines could stimulate competent cells to exchange DNA in order to adapt to the environment In DHABs, is there free DNA? In DHABs, are there natural competent bacteria? Could brines stimulate the competence in these strains? Could brines enhance natural transformation and HGT?

250ng 50ng 10ng 2ng 1min 6hours 1day 2days 8days DNA stability in brines DNA is detectable in Discovery brine for at least 1 day: after exposure to brines DNA maintains the trasforming ability?

Trasformation experiments donor plasmid pZR80: broad host range plasmid confers resistance to ampicillin and kanamycine host strains: isolated strains of the  -proteobacteria group Washed cells Ap S, Km S Incubation with pZR80 Plating on Ap and Km Screening for Ap R Km R colonies Natural competent strain

5D18B Alteromonas macleodiiHalomonas meridiana NaCl 5%0.E+003.E-08 PCB0.E+008.E-10 OTB0.E+001.E-08 filter1.E-09 brine0.E+008.E-05 Trasformation experiments Natural competent strain? No plasmid could be detected in Ap/Km resistant strains