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Please note: This is a scientific communication for personal use only. All data is an intellectual property of Ana I. S. Esteves. Please do not copy or use without consent. For more info or to obtain permission, please email me at aidsesteves@gmail.com Thank you. Marine sponges

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  1. Please note: This is a scientific communication for personal use only. All data is an intellectual property of Ana I. S. Esteves. Please do not copy or use without consent. For more info or to obtain permission, please email me at aidsesteves@gmail.com Thank you

  2. Marine sponges and their symbionts: soulmates or partners by chance? Ana I. S. Esteves Microbial Ecology and Evolution Centre of Marine Sciences of Algarve, Portugal IAAC Jena, 28th September 2011

  3. Marine Sponges Ancient multicellular animals Benthic filter feeders No cell differentiation Totipotency High gene density Diverse morphologies and habitats Extremely sociable! Essentially chemically defended IAAC Jena, 28th September 2011

  4. Marine Sponge Biotechnology Sponges are the most prolific producers of new compounds • Diverse applications: • Pharmaceuticals • Antivirals, antitumorals, antibiotics, analgesics, anti-inflamatories… • Technology • Anti-fouling, biomaterials, bioremediation… Scale-up problem Bluntet. al. 2009 Nat. Prod. Rep. 26, 170-244 Host or symbiont? IAAC Jena, 28th September 2011

  5. Sponge hidden microbial world Microbes can make up to 40% of the sponge volume Less than 1% has been cultivated Dense, diverse and specific microbial communities Beneficial to host – carbon fixation, chemical defense… Taylor et. Al 2007 Microbiol. Mol. Biol. Rev. 71(2): 295-347 IAAC Jena, 28th September 2011

  6. „Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“ Objectives: Determine bacterial diversity in marine sponges of the family Irciniidae Reveal the interplay between the host and its biogeographicalsettingin shaping the structure of these communities Unveil antimicrobial properties of culturable bacteria from Irciniidae specimens AZO ALG MAD Sarcotragus spinosulus Ircinia variabilis Algarve IAAC Jena, 28th September 2011

  7. „Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“ Methodology Culture dependent approach Culture independent approach IAAC Jena, 28th September 2011

  8. 454 Pyrosequencing -DNA is cut in fragments (or specific PCR) and single stranded -Each fragment adheres to one bead -Fragment is amplified 1 bead = millions of copies -Each bead goes into one well -Sequencing takes place • Advantages over DGGE: • You get sequences and not bands • You can know what each sequence is • High Throughput: no need for cut-and-clone bands • -400k sequences per run • (in our case: 6000 – 9000 seqs/sample) IAAC Jena, 28th September 2011

  9. „Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“ A glimpse on pyrosequecing results… sponge 4 dominant phyla Acidobacteria and Actinobacteria enriched sponges homogenate Cyanobacteria and Flavobacteria “de-selected” in sponge vs seawater Plate washing IAAC Jena, 28th September 2011

  10. „Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“ + Irciniidae in a bottle: culturable bacteria from Ircinia sp.and Sarcotragus sp. 25 mL CMFASW 2.5 g Marine Agar ̴3 days DNA extraction Centrifuge 10.000 g 5 min Homogenate 25 CFU/sponge sample 10 Sarcotragus sp. 4 Ircinia sp. 327 isolates 155 ≠ genotypes 16S rDNA PCR BOX-PCR profiling IAAC Jena, 28th September 2011 Marine Broth

  11. 16S rDNA Diversity DNA of isolated bacteria PCR amplification with primers specific for (bacterial) 16S Sequence electropherogram Check in agarose gel RDP Classifier ARB NCBI BLAST IAAC Jena, 28th September 2011

  12. BOX-PCR Genotyping Same 16S rDNA sequence DOES NOT MEAN same strain! BOX element -154 bp highly conserved repetitive DNA sequence, located in distinct, intergenic positions around the genome of bacteria Bac. B Bac. A F5 F3 F2 F4 F1 Agarose gel PCR with specific (single) primer IAAC Jena, 28th September 2011

  13. BOX-PCR Genotyping Microbulbifer sp. BOX-PCR Fingerprint Gelcompar 17 isolates 4 different genotypes IAAC Jena, 28th September 2011

  14. „Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“ Irciniidae in a bottle: culturable bacteria from Ircinia sp.and Sarcotragus sp. Alpha Gamma Culturable Bacteria Diversity -Dominance of Proteobacteria -Reproducible both for Ircinia sp. and Sarcotragus sp. -Pseudovibrio sp., Vibrio sp. and Ruegeria sp. most abundant and transversal to both sponge species – core culturable community? -Considerable higher diversity in Sarcotragus sp. IAAC Jena, 28th September 2011

  15. „Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“ Irciniidae in a bottle: culturable bacteria from Ircinia sp.and Sarcotragus sp. 12 potential new species Related to Amphritea, Aquimarina, Endozoicomonas, Pseudovibrio and Pseudoalteromonas 1 potential new genus Related to Endozoicomonas Ircinia variabilis Sarcotragus spinosulus Sarcotragus spinosulus 16S rDNA phylogeny for Aquimarina sp. (ARB software; forward sequences only) 16S rDNA phylogeny for Endozoicomonas sp. (ARB software; forward sequences only) IAAC Jena, 28th September 2011

  16. „Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“ Irciniidae in a bottle: culturable bacteria from Ircinia sp.and Sarcotragus sp. √ Biotech Potential Polyketide Synthase (PKS) & Non-ribosomal Syntethase (NRPS) Genes Pseudovibrio: 98% PKS+ 48% NRPS+ 20% NRPS++ Ruegeria: 25% PKS+ 11% NRPS+ Vibrio: 31% PKS+ 3% NRPS+ Aquimarina: 92% PKS+ 51% homology with type I PKS from marine Mycobacterium sp. IAAC Jena, 28th September 2011

  17. „Unlocking the bacterial diversity encrypted in the metagenomes of marine sponges“ Irciniidae in a bottle: culturable bacteria from Ircinia sp.and Sarcotragus sp. Whole-cell Antagonistic Assay Competition of bacterial strains by the use of bioactive metabolites Low activity No reproducibility Rethink assay! IAAC Jena, 28th September 2011

  18. Taking one step further… Functionality Deciphering the codes of communication between marine sponges and their symbionts: an integrative metabolomics-transcriptomics approach IAAC Jena, 28th September 2011

  19. “Deciphering the codes of communication between marine sponges and their symbionts” IAAC Jena, 28th September 2011

  20. “Deciphering the codes of communication between marine sponges and their symbionts” IAAC Jena, 28th September 2011

  21. “Deciphering the codes of communication between marine sponges and their symbionts” Fresh from the lab: Biofilm formation IAAC Jena, 28th September 2011

  22. “Deciphering the codes of communication between marine sponges and their symbionts” What is a biofilm? Matrix of Extracellular Polymeric Substances (EPS) – polysaccharides, protein and DNA – that surrounds and serves as support and protection for bacteria, promotes cell adhesion and difusion of chemical signals . IAAC Jena, 28th September 2011

  23. “Deciphering the codes of communication between marine sponges and their symbionts” Why study biofilm formation? “Bacterial biofilm formation can be related to quorum-sensing, antibiotic resistance and adhesive properties.” StepanoviĆ, S., et al. (2007), APMIS 115(8): 891-899 “Bacteria growing in a surface-attached biofilm have increased production of chemical defense mechanisms as compared to planktonic growth. This may represent an adaptive response to compensate for the loss of the ability to escape predation when competing under stressful conditions such as those found inside the sponges.” Santos, O.C.S., et al. (2010), Research in Microbiology, 161(7): 604-612 IAAC Jena, 28th September 2011

  24. “Deciphering the codes of communication between marine sponges and their symbionts” Biofilm producing bacteria adhere to wells, planktonic bacteria stay in suspension Adherent cells stick to wells, planktonic bacteria are washed away Adherent bacteria nuclei are stained; non-binding dye will be washed away Concentration of dye is proportional to number of cells, which in turn is proportional to quantity of biofilm IAAC Jena, 28th September 2011

  25. “Deciphering the codes of communication between marine sponges and their symbionts” Protocol: 180 uL Bacteria culture 200 uL Bacteria culture • Results: • 69% of isolates produce biofilm • 8 strong biofilm producers (without sponge extract): Microccocus sp., Pseudovibrio sp. and Ruegeria sp. • 43% biofilm enhancement with sponge extract; strongly enhanced: Microccocus sp. (Mc110) and Ruegeria sp. (Rg351) • 8% slight biofilm inhibition with sponge extract: Aquimarina sp., Pseudovibrio sp. and Vibrio sp. • No correlation between biofilm formation and cell growth (OD 620 nm) Protocol adaptation: 20 uL Sponge Extract IAAC Jena, 28th September 2011

  26. “Deciphering the codes of communication between marine sponges and their symbionts” To be continued… • Minimum sponge extract concentration needed to enhance biofilm • Response of biofilm formation to synthetic AHLs • Sponge extract biofilm assay guided fractionation • Changes in the exo-metabolome of bacteria in response to sponge extract • AHL screening in sponge isolated bacteria • Preliminary chemotaxis screening IAAC Jena, 28th September 2011

  27. The Microbial Ecology and Evolution Research Group! Rodrigo Costa, the Boss! Cristiane Cassiolato, PhD student ? Port wine!!! Yeah!!!  IAAC Jena, 28th September 2011

  28. Acknowledgements €€€ Portuguese Science and Technology Foundation (FCT) €€€€ Project PTDC/MAR/101431/2008 CCMAR/FCT: Post-doctoral Fellowship CCMAR/BPD/0002/2011 Our divers/sponge taxonomists: Jorge Gonçalves & Group (CCMAR) Joana Xavier and Francisco Pires (University of Azores) IAAC – University of Jena Thomas Wichard Dankeschön! IAAC Jena, 28th September 2011

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