Taxonomy of R pachyptila Riftia pachyptila according to Greg Rouse Riftia pachyptila The vestimentiferan world map http www pbs org wgbh nova abyss life tubewormsans html Distribution of deep water hydrothermal vent communities Vent fauna 1 Working with vent animals Symbiont data Trophosome tissue 16 animal s net weight bacterial cell densities 1010 1011 g Intracellular symbionts contained in bacteriocytes Sulfur oxidizing gamma proteobacteria Net fixation of CO2 Little genetic variability among cells within a single host Little genetic variability across geographic range Have never been cultured 2 Hemoglobin characteristics Riftia plume a Vascular and coelomic hemoglobin b Hemoglobin can bind oxygen sulfide nitrate c Duties Transport of chemicals to the trophosome and prevention of sulfide poisoning Hemoglobin biochemistry a vascular 2 Hb large 3 6 mDalton arranged in hexagonal bilayer and small 400 kDalton coelomic different small 400 kDalton b Binding of sulfide supposed to be through cystein bonds however experiments not reproducible Three PNAS papers c Mechanisms for nitrate binding unknown Key Questions Nutrient Acquisition Nitrogen assimilation Carbon incorporation Nature of Association Symbiont transmission Adaptation to symbiotic life Genes lost or gained Metabolic characteristics Requirements for culturing Symbiont purification by Percoll gradient Transfer product Gradient Symbionts 3 Transfer product s Pellet 3 phosphoglyceric acid malate succinate sugars alanine aspartate Supernatant succinate glutamate Symbiont purification by Percoll gradient SUNLIGHT VENTS SULFIDE Plants photosynthetic microorganisms etc Oxygen Chemosynthetic microorganisms free living and symbiotic Gradient Animals heterotrophic organisms Animals heterotrophic organisms Symbionts Proteomics 2132 ORFs predicted to be cytoplasmic 255 ORFs predicted to be periplasmic 2387 theoretical soluble proteins 1000 spots analyzed 500 individual proteins identified 4 Functional Genome Analysis Membrane proteome proteome reference maps II Comparative Proteomics analysis of the cellular response to varying environmental conditions reconstruction of basic metabolic pathways detection of regulatory networks I Mastergel Classical membrane protein isolation and isolation of protein associated to sulfur globule membranes 822 ORFs predicted to have TMHs 383 TMH containing proteins identified TMH trans membrane helix II Comparative Proteomics comparison of bacterial protein patterns light trophosome dark trophosome Riftia trophosome tissue from freshly dissected worms binocular view A Light green B Green C Dark green In light trophosomes the natural green color of the host cell layer surrounding the bacteriocytes is overlaid by the light color of the elemental sulfur globules fused image n 3 Dark colored Light colored Changes in protein concentrations in light and dark green trophosome Protein Ratio Sulfite reductase DsrA 18 9 APS reductase ApsA 16 1 Rubisco CbbM 3 24 1 73 Alkylhydroperoxide reductase AhpC 12 0 Glycolate oxidase GlcD 9 14 Succinyl CoA synthetase SucD 8 51 ketoglutarate ferredoxin oxidoreductase KorG 4 18 SucD kDa 80 CcbM Heterodisulfite reductase HdrA Symbiont purification by Percoll gradient MW HdrA DsrL ApsA fused image n 3 KorA AcnA 50 DsrA KorB GlcD 30 Gradient 20 KorG Symbionts AhpC light green pI 7 0 dark green 10 www decodon de pI 4 0 5 Riftia pachyptila Metagenome Whole Genome Shotgun Results Estimated genome size 3 6 4 0 Mb Base pairs sequenced 44 897 623 phred 10 11X coverage Is there more than one symbiont species 103 Contigs Diversity by metagenome All 16S rRNA sequences in the metagenome are 100 identical SNP Sequence comparison for pyruvate ferredoxin oxidoreductase of Riftia and Tevnia symbiont Contig 1135 0 5 polymorphic Sequence comparison for aconitase of Riftia and Tevnia symbion Some results originating from sequencing 6 Flagella Enzymes of nitrate assimilation in Riftia symbionts Respiratory nitrate reductase alpha beta gamma chain Nitrite reductase small and large subunit Nitric oxide reductase several components No nitrous oxide reductase No assimilatory nitrate reductase Reductive TCA cycle in Riftia pachyptila Pyruvate carboxylase Pyruvate CoA pyc 6 4 1 1 CO2 Oxaloacetate fumarate reductase SdhAC 1 3 99 1 L Malate ATP 6 2 1 18 Citrate H2O NAD cis Aconitate H2O H2O aconitase AcnA 4 2 1 3 aconitase AcnA What effects could two pathways for carbon fixation have 4 2 1 3 Fumarate NADH2 Isocitrate NAD P NAD NAD P H2 Succinate ATP Oxalsuccinate CoA succinyl CoAsynthetase SucCD 6 2 1 5 ADP Pi 1 citryl CoA synthetase CcsB CoA Citryl CoA NADH2 Mdh 1 1 1 37 FumB 4 2 1 2 PorAG 1 2 7 1 Acetyl CoA malatedehydrogenase fumarase pyruvate ferredoxin oxidoreductase CO2 ADP Pi CO2 Ketoglutarate Succinyl CoA NADH2 NAD CO2 CoA isocitratedehydrogenase Icd 1 1 1 42 isocitratedehydrogenase Icd 1 1 1 42 ketoglutarateferredoxinoxidoreductase KorB 1 2 7 3 Carbon Isotope ratios CO2 uptake in live Riftia pachyptila Hydroxypropionate Reductive TCACalvin BensonReductive acetyl CoA Pathway Cycle Cycle Pathway Chloroflexus Chlorobium Thiomicrospira Desulfobacterium 0 10 Riftia pachyptila 20 30 40 13C Slide provided by Peter Girguis 7 The early life of Riftia How do they colonize new vent areas How do they obtain symbionts Biochemical composition and buoyancy of Riftia eggs Early development of Riftia larvae grown in pressure chambers a 1 cell 1 day b 2 cell 3 days c 8 cell 7 days d 128 cell 14 days e 34 day larva with ciliary bands Development and respiration rates of Riftia embryos a Pressure vessels open in situ closed symbols b 32 64 cells open 4 16 cells closed Dispersal potential of larvae of Riftia pachyptila a Current velocities represented by arrows showing compass heading and velocity b Plan view of the trajectory of a water parcel with its origin star at the location of the current meter and its path calculated from hourly averaged velocities filled circles denote positions at weekly intervals grey line denotes the ridge axis c Along axis distances traveled by larvae with a selected 26 d lifespan released at hourly intervals in the 9 N current regime 39 remained within 25 km of the ridge axis The maximum dispersal distance for a survivor with this 26 d lifespan was 103 km SSE How are symbionts acquired 8 Free living lifestyle Vestimentiferans settled on Baby Traps Acquisition of symbionts Tubeworm larva Early Development b brain mo mouth fg mg hg gut tr trophosome dv vv blood vessels or opturaculum pr