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CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare

Daniele Daffonchio Tullio Brusa Sara Borin Enrica Canzi. Alessandro Favini Lorenzo Brusetti Claudia Sorlini. Partner 1c. CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare. DISTAM Università degli Studi di Milano. WP3

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CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare

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  1. Daniele Daffonchio Tullio Brusa Sara Borin Enrica Canzi Alessandro Favini Lorenzo Brusetti Claudia Sorlini Partner 1c CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare DISTAM Università degli Studi di Milano

  2. WP3 “Analysis of the species diversity, community structure and phylogeny of micro-organisms and meiofauna in the Mediterranean deep hypersaline anoxic basins (DHABs)” • 1st year objectives: • development of methods for the analysis of DHABs prokaryotic communities • characterisation of micro-organisms abundance in DHABs samples • characterisation of microbial diversity in the 4 sampled basins • description of Urania seawater-brine chemocline

  3. 15ml stained with DAPI and filtered on 0.22m filters 50ml incubated with substrates and BrdU and filtered on 0.22m filters Different layers stored at -20°C 1-5l filtered on 0.22m filters ? Microscopic microbial counts DNA extraction DNA extraction DNA extraction and capture ? 1st year activity Microbial abundance and diversity Microbial abundance and diversity Microbial functional diversity Development of methods for the analysis of DHABs bacterial communities seawater water/brine interface brines DHABs samples brine/sed interface sediment on board Urania 2001 cruise

  4. DAPI cell counts cells/ml of sample W 1500m L’Atalante Discovery Bannock Urania 2,8 104 W 2500m Water column 1500m 4,3 104 2500m W 3300m 3,9 104 2,3 104 3300m Water/brines interface 6,1 105 3,4 106 1,2 106 1 106 WB 1,9 104 1,5 105 7,8 104 4,7 104 brines B 3500m

  5. DNA extraction yields g DNA/l of sample W 1500m L’Atalante Discovery Bannock Urania 0,2 W 2500m Water column 1500m 0,2 2500m W 3300m 0,2 0,1 3300m Water/brines interface 20 20 11 15 WB 0? 0,5 0,6 0,1 brines B 3500m

  6. Brines act as “particle traps” and are an enrichment phase for bacteria Are the microbial communities in the 4 basins inhabited by specific bacterial communities, different from the upper seawater?

  7. ARDRA • restriction enzymes ITS-HHP SSCP Number length sequence Polymorphisms in Denaturation or digestion of one phosphorilated strand Heteroduplex bands Different bands for different sequences Homoduplex bands Molecular typing of brines and water/brine interface microbial communities based on the ribosomal operon tDNA 16S rDNA 23S rDNA 5S

  8. ITS-HHP eubacteria ARDRA eubacteria SSCP archaea interface interface interface interface interface interface interface interface brines interfaces brine brine brine brine brine A B U D U U A A B D B A U D B A U basin

  9. Urania Discovery I I L’Atalante B B I B Bannock I PC 2 (10% of the variability) Upper seawater column PC 1 (29,5% of the variability) Diversity of eubacterial communities along the depth profile: Principal Component Analysis

  10. Unidentifyed euryrachaeota Halophilic archaea Methanococcales Thermococcales 3 Characterization of brines Archaea composition Construction of libraries of a 150 bp fragment from DNA extracted from Urania and L’atalante brines with primers specific for Archaea 16S (V6 region)

  11. Urania Water/brine interface: is the salinity gradient inhabited by different microbial communities? Brines Conductivity (salinity) Seawater 3520m 3523m

  12. 10L Niskin bottle n°3 10L Niskin bottle n°4 WATER URANIA INTERFACE depth BRINES

  13. 10th litre …. 4th litre DNA extraction and microbial community molecular characterisation 3rd litre 2nd litre 1st litre Water/brine interface: is the salinity gradient inhabited by different microbial communities? 10 L Niskin bottle

  14. Water/brine interface: the salinity gradient does not substantially influence the diversity of eubacterial communities Niskin 3 Niskin 4 Niskin 3 Niskin 4 ITS-HHP eubacteria ARDRA eubacteria

  15. Cluster analysis of ARDRA fingerprinting similarity Niskin 4 Niskin 3

  16. Real Time quantification of Archaeal and Eubacterial 16S genes On samples from the Urania basin have been applied Primers and TaqMan probes specific for a region of the 16S gene: • universal for prokariotes • universal for archaea

  17. eubacteria archaea Along the salinity gradient there are quantitative differences among archaea/eubacteria populations

  18. sample Immunocapture BrdU-labelled DNA Community structure analysis of the actively growing bacteria Characterization of bacterial functional diversity in the Discovery basin Bromodeoxyuridine BrdU samples substrate • 3300m depth water • water/brine interface • brines + O2 Incubation Filtration DNA extraction - O2 On board 1st year substrates • propionate • aminoacids • citrate • catechol • oil

  19. donor MATING host Screening for: Hg resistance NaCl tolerance Results: no transconiugants “Fishing” of functional genes in the metagenomic DNA Plasmids donor: cells recovered filtrating Discovery water/brines interface Plasmids exogenous isolation Plasmids hosts: Pseudomonas putida Agrobacterium tumefaciens Ralstonia eutropha Escherichia coli

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