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  1. ULIXES(EU FP7 Project – GA-266473)“Unravelling and expLoiting MedIterranean Sea microbial diversity and ecology for XEnobiotics’ and pollutantS’ clean up”Daniele DaffonchioScientific Coordinator, Partner 1 - DiSTAM, University of Milan, Italy1ST YEAR PROJECT MEETINGCapo Granitola, Sicily (Italy) – 21-23 January 2012 Project scientific advancements at UMIL

  2. Presentation outline • Objectives • Achievements • To be done.. 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  3. UMIL CONTRIBUTION TO WORK PACKAGES • WP1- Management • WP2- Sampling activities • WP3- Definition of a map of microbial diversity in Med polluted sites • WP4- Establishment of a collection of isolates and mixed cultures with catabolic capabilities • WP7, WP8- Develop processes/in situ testing of reductive dehalogenation of chlorinated compounds • WP9- Results dissemination 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  4. WP2: SAMPLING AND CHARACTERISATION OF THE MED SEA SITES • Polluted sites: • Transects against the oil tanker routes in the whole Med (water) • Sunk tanker Haven, 50000 tons crude oil spilled 33-83 m off Genoa coast (sediments) • Venice lagoon & Augusta bay (sediments) • Natural deep-sea hydrocarbon seepages: • Olimpi, Amsterdam, Kula mud volcanoes (water and sediments) • Urania hypersaline deep anoxic basin mud pit (sediments) and other DHALs 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  5. WP3: MICROBIAL DIVERSITY EXPLORATION IN THE MED SEA SITES • Based on: • extraction and analyses of total DNA and RNA from water and sediment samples • microscopic counts (DAPI staining) • Molecular fingerprinting and Real Time PCR quantification methods to estimate: • the overall diversity of colonizing microbial populations (molecular clocks i.e. ribosomal operon) • catabolic diversity and potential (specific known degrading genes) 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  6. WP4:ESTABLISHMENT OF COLLECTIONS OF PROKARYOTE ISOLATES AND METABOLITES SEAWATER • Hydrocarbons as unique C source • 1,2-DCA and electron donors ENRICHMENT CULTURES SEDIMENT Collection of isolates Strain identification Mixed cultures Molecular identification of species • Physiological characterisation • Characterisation of degrading genes WP7, WP8: establishing and testing new products by pilot bioremediation treatments 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  7. Presentation outline • Objectives • Achievements • To be done.. 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  8. WP2: SAMPLING CRUISES ULIXES-M11-1/4-7 CRUISE, 5th- 28th April 2011 5-liters water samples have been collected from 46 sites, most of them located along the transects of oil tanker in the whole Med and especially in correspondence of sites 17a-b-c 17a 17b 17c ULIXES CRUISE, 9th- 23th September 2011 Water and sediment samples have been collected from different deep hypersaline anoxic basins including Tyro, Kryos, Urania Mud Pit and from Napoli mud volcano. 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  9. WP3: DAPI COUNTS ULIXES-M11-1/4-7 CRUISE, 5-28 April 2011 309 311 313 292 293 294 329 331 333 298 299 302 SHALLOW WATER SAMPLES ALONG OIL TANKER ROUTES SHALLOW AND DEEP (103 m) WATER SAMPLES IN THE LEVANTINE BASIN Cells ml-1 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  10. 309 311 313 In progress 329 331 333 298 299 302 292 293 294 WP3: DIVERSITY DATABASE OF MICROBIAL SPECIES (D3.1) ULIXES-M11-1/4-7 CRUISE, 5-28 April 2011 Augusta Bay Messina polluted sand El-Max District, Alexandria (Egypt) HAVEN 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  11. WP3: DGGE 309 311 313 Med Sea Aqaba 329 331 333 MS11 PH11 FR11 298 299 302 O11 294 331 292 299 309 333 293 298 302 313 329 311 292 293 294 40 % denaturinggradient 60 % 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  12. WP3: DGGE Aqaba sediment Aqaba water El-Max District, Alexandria (W1 and W2) MS11- MS33 FR11- FR31 O11- O33 PH11- PH23 PH23-c O33-B MS33 MS11 FR31 PH11 FR11 Me-a AU-a HS-b O11 W1 W2 Messina polluted Sand (Me-a) 40 % HAVEN (HS-b) Augusta Bay (AU-a) denaturinggradient 60 % 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  13. WP3: DGGE • Detection of 16S rRNA partial sequences previously described in similar environments • Detection of 16S rRNA partial sequences associated to : • - marine oil-polluted sediments • - nitrate-treated oil production facility • - petroleum-aromatic-contaminated tropical marine environments • - Deepwater Horizon oil spill 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  14. WP3: DGGE dsrB GENE MS33 HS-b Me-a AU-a S1 Messina polluted Sand (Me-a) El-Max District, Alexandria (S1) HAVEN (HS-b) Augusta Bay (AU-a) MS33 PCR primer set dsr-GC 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  15. WP4: ESTABLISHMENT OF CULTURE COLLECTION Set up of enrichment cultures and pure strain isolation and characterization Collection 1: hydrocarbon degrading prokaryotes Collection 2: halogenated compounds degrading prokaryotes WP4: CULTURE COLLECTION 1 Water samples have been collected from a subset of stations during ULIXES-M11-1/4-7 cruise to set-up enrichment of hydrocarbon degrading bacteria (HDB). • 4 stations in the Levantine basins • 4 stations in the Alboran Sea • 1 stations outside Gibraltar strait Enrichment on ONR7a + gasoil Isolation of pure cultures (~200 strains) 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  16. WP4: COLLECTION 1, STRAINS IDENTIFICATION 333 334 Levantine basin 339 Alboran Sea Alcanivorax diesololei Alcanivorax jadensis Alcanivorax venustensis Strain identification will be completed in two weeks 329 331 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  17. WP4: CULTURE COLLECTION 2 Set-up of anaerobic microcosms from sediments for enrichment of DCA-degrading bacteria • Messina polluted Sand (Me-a), Augusta Bay (AU-a), HAVEN (HS-b), Aqaba (MS33), El-Max District Alexandria (S1) • PCR test for the presence of dcaA genes, molecular marker of dehalorespiring microbial consortia (in progress) • Conditions set up for evaluation of DCA degradation by headspace gas chromatography Mineral medium with NaCl 3.5% Na-lactate 0.2 % (electron donor) Vitamin B12 (corrinoid cofactor of dehalogenases) 1,2-DCA 50 mg/l HS-b - DCA + DCA PCR primer set PceAFor1/DcaB rev (Marzorati et al., 2010) Control + To date… GC-FID, Carbopack column, injector T 200°C, oven T 160°C, carrier gas N2 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  18. WP4: CULTURE COLLECTION 2 • Reactivation of microcosms involved in 1,2-dichloroethane (1,2-DCA) reductive declorination • progressive adaptation to salinity values similar to seawater • monitoring microbial community structure reactivated no salt 0.25% NaCl reactivated no salt 0.25% NaCl D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D3 Sequencing in progress 16s rRNA gene dsrB gene 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  19. WP4: DCA DEGRADING CULTURE “Canale Brentella” sediments 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  20. Presentation outline • Objectives • Achievements • To be done.. 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  21. Next goals to be achieved.. WP3: • ARISA fingerprinting on metagenome extracted from sea water and sediments collected: • Along oil tanker routes in the Med Sea • From polluted sites including Haven shipwreck, Augusta bay, Aqaba Gulf, El-Max district, ………. all the other sites WP4: • Monitoring of DCA enrichments obtained from anaerobic sediments collected in polluted sites by DCA consumption measurements • Gradual adaptation of microcosms to higher salinity values 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  22. RDH GENE ASSOCIATED TO PCBs RD +Vancomycin & Ampicillin +Vancomycin +Ampicillin NO PCB PCB PCB NO PCB PCB PCB NO PCB NO PCB 1500 bp Detection of reductive dehalogenase (RDH) genes in PCB-spiked and PCB-free cultures that were not amended with electron donors 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  23. 359 10 415 Reductive deahlogenase gene: schematic diagram Schematic diagram of gene fragments amplified from Dehalococcoides sp. strains with the primer pair RRF2-B1R. rdhA represents the open reading frame encoding the catalytically active reductive dehalogenase, and rdhB is the open reading frame encoding a small hydrophobic B protein. Tat signal, twin-arginine translocation signal sequence including the RRXFXK consensus motif. (HOLSCHER et al., 2004) Deduced amino acid sequence of the putative dehalogenase PCB-RDH iron-sulfur binding motif II twin-arginine motif iron-sulfur binding motif I Alignment of the deduced amino acid sequence of the putative dehalogenase PCB-RDH from this study (Acesion number of the nucleotide sequenceFR839675). The aminoacid characterizing the twin-arginine motif (first sequence block) and the two iron-sulfur binding motifs (second and third sequence blocks) are marked in light grey. Sequence abreviation, species, and protein identity are as follows: RDH BAV1, Dehalococcoides sp.BAV1, ABQ16871; RDH VS, Dehalococcoides sp. VS, ACZ61269. “Canale Brentella” sediments 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012

  24. Phylogenetic positioning of the nucleotide sequence of the putative reductive dehalogenase PCB-RDH from this study with other related reductive dehalogenase sequences PCB_RDH BAV1(295198..296688) BAV1_RdhB4 (AY553225) CBDB1-RdhB12 (AY374240) Reductive dehalogenase gene detected in “Canale Brentella” sediments CBDB1-RdhA13 (AY374241) CBDB1-RdhB14 (AY374242) Holscher,T., et al., Multiple nonidentical reductive-dehalogenase-homologous genes are common in Dehalococcoides. Appl. Environ. Microbiol. 70 (9), 5290-5297 (2004) BAV1_RdhB1 (AY553222) CBDB1-RdhB3 (AY374231) CBDB1-RdhB4 (AY374232) Dehalococcoides sp. FtL-RDase-1638 (EU137844) Santoh,K, et al., Detection of a Bacterial Group within the Phylum Chloroflexi and Reductive-Dehalogenase-HomologousGenes in Pentachlorobenzene-DechlorinatingEstuarineSedimentfrom the Arakawa River, Japan. MicrobesEnviron. 21, 154-162 (2006) BAV1_RdhA7 (AY553228 ) CBDB1-RdhA1 (AY374229) CBDB1-RdhA2 (AY374230) Dehalococcoides sp. FtL-RDase-1638 (EU137843) CBDB1-RdhA5 (AY374233) McMurdie,P.J., et al., Localized plasticity in the streamlined genomes of vinyl chloride respiring Dehalococcoides. PLoS Genet. 5 (11), E1000714 (2009) CBDB1-RdhB6 (AY374234) BAV1_RdhB3 (AY553224) CBDB1-RdhB7 (AY374235) Lee,P.K. et al., Quantifying genes and transcripts to assess the in situ physiology of Dehalococcoides spp. in a trichloroethene-contaminated groundwater site.Appl. Environ. Microbiol. 74 (9), 2728-2739 (2008) RDH6 (AB251618) BAV1_RdhA6 (AY553223) RDH4 (AB251617) RDH 1 (AB251615) BAV1 _RDH5 (AY553226) Ritalahti,K.M., et al., Genetic identification of putative reductive dehalogenase genes from a 1,2-dichloropropane dechlorinating enrichment culture derived from the King Salmon River, Alaska. Unpublished CBDB1-RdhA10 (AY374238 ) CBDB1-RdhB8 (AY374236) CBDB1-RdhA9 (AY374237) CBDB1-RdhB16 (AY374244) CBDB1-RdhB15 (AY374243) The phylogenetic analysis shows that there is no correlation between the RDH sequences and the specificity of the substrate or the taxonomic affiliation Kube,M., et al., J Genome Sequence of Dehalococcoides Strain CBDB1: Specialization to Respiration with Chlorinated Compounds. Nature Biotech 23 (10), 1269-1273 (2005) CBDB1-RdhB11 (AY374239) KSRdA09 putative rdhA gene(EU137843) BAV1 vinyl-chloride-reductive-dehA (AY563562) BAV1_RdhA6 (AY553227) Dehalococcoides sp. FtL-RDase-bvcA (EU137841) “Canale Brentella” sediments Dehalococcoides sp. FtL-RDase-vcrA(EU137842) putative rdhA of Dehal. sp.VS (ACZ61269) CBDB1-putative RDH cdbdA80 (AJ965256) 1st YEAR PROJECT MEETING, Sicily, 21-23 January 2012 CBDB1-putative RDH cdbdA84 (CAI82345) RDH3 (AB251616)

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