#METABOLOMICS WORKBENCH TruxalCarlson_20201104_100140 DATATRACK_ID:2225 STUDY_ID:ST001524 ANALYSIS_ID:AN002545 PROJECT_ID:PR001025 VERSION 1 CREATED_ON November 5, 2020, 11:44 am #PROJECT PR:PROJECT_TITLE Prochlorococcus extracellular vesicles: Molecular composition and adsorption to PR:PROJECT_TITLE diverse microbial cells PR:PROJECT_TYPE Marine Metabolomics PR:PROJECT_SUMMARY Extracellular vesicles are small (~50–200 nm diameter) membrane-bound PR:PROJECT_SUMMARY structures released by cells from all domains of life. While extremely abundant PR:PROJECT_SUMMARY in the oceans, our understanding of their functions, both for cells and the PR:PROJECT_SUMMARY emergent ecosystem, is in its infancy. To advance this understanding, we PR:PROJECT_SUMMARY analyzed the lipid, metabolite, and protein content of vesicles produced by two PR:PROJECT_SUMMARY strains of the most abundant phytoplankton cell in the ocean, the cyanobacterium PR:PROJECT_SUMMARY Prochlorococcus. We show that Prochlorococcus exports an enormous array of PR:PROJECT_SUMMARY cellular compounds into their surroundings via extracellular vesicles. The PR:PROJECT_SUMMARY vesicles produced by the two different strains contained some materials in PR:PROJECT_SUMMARY common, but also displayed numerous strain-specific differences, reflecting PR:PROJECT_SUMMARY functional complexity within natural vesicle populations. Prochlorococcus PR:PROJECT_SUMMARY vesicles contain active enzymes, indicating that they can mediate PR:PROJECT_SUMMARY biogeochemically relevant extracellular reactions in the wild. Interaction PR:PROJECT_SUMMARY assays demonstrate that vesicles from Prochlorococcus and multiple genera of PR:PROJECT_SUMMARY heterotrophic bacteria can associate with other marine microbes, including PR:PROJECT_SUMMARY Pelagibacter, the most abundant heterotrophic group in the oceans. Our PR:PROJECT_SUMMARY observations suggest that vesicles may play diverse functional roles in the PR:PROJECT_SUMMARY oceans, including but not limited to mediating energy and nutrient transfers, PR:PROJECT_SUMMARY catalyzing extracellular biochemical reactions, and mitigating toxicity of PR:PROJECT_SUMMARY reactive oxygen species. These findings further indicate that a portion of the PR:PROJECT_SUMMARY ‘dissolved’ compounds in the oceans are not truly dissolved, but are instead PR:PROJECT_SUMMARY packaged within locally structured, colloidal vesicles. PR:INSTITUTE University of Washington PR:DEPARTMENT Oceanography PR:LABORATORY Ingalls Lab PR:LAST_NAME Carlson PR:FIRST_NAME Laura PR:ADDRESS 1501 NE Boat Street, Marine Science Building, Room G, Seattle, WA 98195 PR:EMAIL truxal@uw.edu PR:PHONE 4125545093 #STUDY ST:STUDY_TITLE Prochlorococcus extracellular vesicles: Molecular composition and adsorption to ST:STUDY_TITLE diverse microbial cells ST:STUDY_TYPE Characterizing the metabolome of Prochlorococcus cells and vesicles ST:STUDY_SUMMARY Extracellular vesicles are small (~50–200 nm diameter) membrane-bound ST:STUDY_SUMMARY structures released by cells from all domains of life. While extremely abundant ST:STUDY_SUMMARY in the oceans, our understanding of their functions, both for cells and the ST:STUDY_SUMMARY emergent ecosystem, is in its infancy. To advance this understanding, we ST:STUDY_SUMMARY analyzed the lipid, metabolite, and protein content of vesicles produced by two ST:STUDY_SUMMARY strains of the most abundant phytoplankton cell in the ocean, the cyanobacterium ST:STUDY_SUMMARY Prochlorococcus. We show that Prochlorococcus exports an enormous array of ST:STUDY_SUMMARY cellular compounds into their surroundings via extracellular vesicles. The ST:STUDY_SUMMARY vesicles produced by the two different strains contained some materials in ST:STUDY_SUMMARY common, but also displayed numerous strain-specific differences, reflecting ST:STUDY_SUMMARY functional complexity within natural vesicle populations. Prochlorococcus ST:STUDY_SUMMARY vesicles contain active enzymes, indicating that they can mediate ST:STUDY_SUMMARY biogeochemically relevant extracellular reactions in the wild. Interaction ST:STUDY_SUMMARY assays demonstrate that vesicles from Prochlorococcus and multiple genera of ST:STUDY_SUMMARY heterotrophic bacteria can associate with other marine microbes, including ST:STUDY_SUMMARY Pelagibacter, the most abundant heterotrophic group in the oceans. Our ST:STUDY_SUMMARY observations suggest that vesicles may play diverse functional roles in the ST:STUDY_SUMMARY oceans, including but not limited to mediating energy and nutrient transfers, ST:STUDY_SUMMARY catalyzing extracellular biochemical reactions, and mitigating toxicity of ST:STUDY_SUMMARY reactive oxygen species. These findings further indicate that a portion of the ST:STUDY_SUMMARY ‘dissolved’ compounds in the oceans are not truly dissolved, but are instead ST:STUDY_SUMMARY packaged within locally structured, colloidal vesicles. ST:INSTITUTE University of Washington ST:DEPARTMENT Oceanography ST:LABORATORY Ingalls Lab ST:LAST_NAME Carlson ST:FIRST_NAME Laura ST:ADDRESS 1501 NE Boat Street, Marine Science Building, Room G, Seattle, WA 98195 ST:EMAIL truxal@uw.edu ST:PHONE 4125545093 #SUBJECT SU:SUBJECT_TYPE Other SU:SUBJECT_SPECIES Prochlorococcus marinus str. MIT 9312;Prochlorococcus marinus MIT9313 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Cell_9312_A Biovolume_extracted:3.69E+09 | Sample Type:Pellet | Cell Type:9312 RAW_FILE_NAME=170124_Smp_Pro9312_A;170124_Smp_Pro9312_A 170128_Smp_Pro9312_A 170128_Smp_Pro9312_A_DCM SUBJECT_SAMPLE_FACTORS - Cell_9312_B Biovolume_extracted:5.04E+09 | Sample Type:Pellet | Cell Type:9312 RAW_FILE_NAME=170124_Smp_Pro9312_B;170124_Smp_Pro9312_B 170128_Smp_Pro9312_B 170128_Smp_Pro9312_B_DCM SUBJECT_SAMPLE_FACTORS - Cell_9312_C Biovolume_extracted:4.95E+09 | Sample Type:Pellet | Cell Type:9312 RAW_FILE_NAME=170124_Smp_Pro9312_C;170124_Smp_Pro9312_C 170128_Smp_Pro9312_C 170128_Smp_Pro9312_C_DCM SUBJECT_SAMPLE_FACTORS - Cell_9313_A Biovolume_extracted:1.98E+09 | Sample Type:Pellet | Cell Type:9313 RAW_FILE_NAME=170124_Smp_Pro9313_A;170124_Smp_Pro9313_A 170128_Smp_Pro9313_A 170128_Smp_Pro9313_A_DCM SUBJECT_SAMPLE_FACTORS - Cell_9313_B Biovolume_extracted:1.27E+09 | Sample Type:Pellet | Cell Type:9313 RAW_FILE_NAME=170124_Smp_Pro9313_B;170124_Smp_Pro9313_B 170128_Smp_Pro9313_B 170128_Smp_Pro9313_B_DCM SUBJECT_SAMPLE_FACTORS - Cell_9313_C Biovolume_extracted:6.87E+09 | Sample Type:Pellet | Cell Type:9313 RAW_FILE_NAME=170124_Smp_Pro9313_C;170124_Smp_Pro9313_C 170128_Smp_Pro9313_C 170128_Smp_Pro9313_C_DCM SUBJECT_SAMPLE_FACTORS - Vesicle_9312_1 Biovolume_extracted:2.55E+07 | Sample Type:Vesicle | Cell Type:9312 RAW_FILE_NAME=170124_Smp_Vesicle9312_1;170124_Smp_Vesicle9312_1 170128_Smp_Vesicle9312_1 170128_Smp_Vesicle9312_1_DCM SUBJECT_SAMPLE_FACTORS - Vesicle_9312_2 Biovolume_extracted:3.71E+07 | Sample Type:Vesicle | Cell Type:9312 RAW_FILE_NAME=170124_Smp_Vesicle9312_2;170124_Smp_Vesicle9312_2 170128_Smp_Vesicle9312_2 170128_Smp_Vesicle9312_2_DCM SUBJECT_SAMPLE_FACTORS - Vesicle_9312_3 Biovolume_extracted:3.72E+07 | Sample Type:Vesicle | Cell Type:9312 RAW_FILE_NAME=170124_Smp_Vesicle9312_3;170124_Smp_Vesicle9312_3 170128_Smp_Vesicle9312_3 170128_Smp_Vesicle9312_3_DCM SUBJECT_SAMPLE_FACTORS - Vesicle_9313_1 Biovolume_extracted:2.50E+08 | Sample Type:Vesicle | Cell Type:9313 RAW_FILE_NAME=170124_Smp_Vesicle9313_1;170124_Smp_Vesicle9313_1 170128_Smp_Vesicle9313_1 170128_Smp_Vesicle9313_1_DCM SUBJECT_SAMPLE_FACTORS - Vesicle_9313_2 Biovolume_extracted:1.97E+08 | Sample Type:Vesicle | Cell Type:9313 RAW_FILE_NAME=170124_Smp_Vesicle9313_2;170124_Smp_Vesicle9313_2 170128_Smp_Vesicle9313_2 170128_Smp_Vesicle9313_2_DCM SUBJECT_SAMPLE_FACTORS - Vesicle_9313_3 Biovolume_extracted:3.24E+08 | Sample Type:Vesicle | Cell Type:9313 RAW_FILE_NAME=170124_Smp_Vesicle9313_3;170124_Smp_Vesicle9313_3 170128_Smp_Vesicle9313_3 170128_Smp_Vesicle9313_3_DCM #COLLECTION CO:COLLECTION_SUMMARY Axenic cultures of Prochlorococcus strain MIT9312 and MIT9313 were grown in CO:COLLECTION_SUMMARY defined artificial AMP1 media supplemented with 10 mM (final concentration) CO:COLLECTION_SUMMARY filter-sterilized sodium bicarbonate. Seven 20 L cultures were grown for each of CO:COLLECTION_SUMMARY the two Prochlorococcus strains, providing three replicates for the lipid and CO:COLLECTION_SUMMARY small metabolite analysis and an additional sample for proteomics analysis. CO:COLLECTION_SUMMARY Strains MIT9312 and MIT9313 are available from the National Center for Marine CO:COLLECTION_SUMMARY Algae and Microbiota. 20 L cultures were grown in polycarbonate carboys CO:COLLECTION_SUMMARY (ThermoFisher Nalgene, Waltham, MA, USA) with gentle stirring (60 rpm), under CO:COLLECTION_SUMMARY constant light flux (10 – 20 µmol Q m -2 s -1 for MIT9313; 30 – 40 µmol Q CO:COLLECTION_SUMMARY m -2 s -1 for MIT9312) at 24°C. Vesicles were collected from 20 L cultures of CO:COLLECTION_SUMMARY Prochlorococcus during mid-to-late exponential growth phase and isolated as CO:COLLECTION_SUMMARY described previously (Biller et al., 2014, Science). Briefly, cultures were CO:COLLECTION_SUMMARY first gravity filtered through a 0.2 µm capsule filter (Polycap 150TC; GE Life CO:COLLECTION_SUMMARY Sciences/Whatman, Maidstone, UK). The filtrate was then concentrated using a 100 CO:COLLECTION_SUMMARY kDa tangential flow filter (Ultrasette with Omega membrane; Pall, Port CO:COLLECTION_SUMMARY Washington, NY, USA) and re-filtered through a 0.2 µm syringe filter. Vesicles CO:COLLECTION_SUMMARY were pelleted from the sample by ultracentrifugation at ~100,000 x g CO:COLLECTION_SUMMARY (Beckman-coulter SW32Ti rotor; 32,000 rpm, 1.5 hrs, 4°C), purified on an CO:COLLECTION_SUMMARY OptiPrep gradient (Biller et al., 2014, Science), then washed and resuspended in CO:COLLECTION_SUMMARY 0.2 µm filtered 1x PBS. CO:SAMPLE_TYPE Cultured Prochlorococcus cells and vesicles CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No treatment - cells and vesicles were cultured according to standard protocols. TR:TREATMENT_SUMMARY We used targeted and untargeted metabolomics to characterize the metabolome of TR:TREATMENT_SUMMARY cells and vesicles. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Each sample was extracted using a modified Bligh-Dyer extraction. Briefly, SP:SAMPLEPREP_SUMMARY quantitative aliquots of cell pellets were transferred into 15 mL teflon SP:SAMPLEPREP_SUMMARY centrifuge tubes containing a mixture of 100 µm and 400 µm silica beads. SP:SAMPLEPREP_SUMMARY Quantitative aliquots of extracellular vesicles were transferred into 24 mL SP:SAMPLEPREP_SUMMARY glass vials and extracted without bead beating. Heavy isotope-labeled internal SP:SAMPLEPREP_SUMMARY standards were added along with ~2 mL of cold aqueous solvent (50:50 SP:SAMPLEPREP_SUMMARY methanol:water) and ~3 mL of cold organic solvent (dichloromethane). The samples SP:SAMPLEPREP_SUMMARY were shaken on a FastPrep-24 Homogenizer for 30 seconds and chilled in a -20 °C SP:SAMPLEPREP_SUMMARY freezer repeatedly for three cycles of bead-beating and a total of 30 minutes of SP:SAMPLEPREP_SUMMARY chilling. The organic and aqueous layers were separated by spinning samples in a SP:SAMPLEPREP_SUMMARY centrifuge at 4,300 rpm for 2 minutes at 4 °C. The aqueous layer was removed to SP:SAMPLEPREP_SUMMARY a new glass centrifuge tube. The remaining organic fraction was rinsed three SP:SAMPLEPREP_SUMMARY more times with additions of 1 to 2 mL of 50:50 methanol:water. All aqueous SP:SAMPLEPREP_SUMMARY rinses were combined for each sample and ~2 mL of cold dichloromethane was added SP:SAMPLEPREP_SUMMARY to the combined aqueous layer. Tubes were shaken and centrifuged at 4,300 rpm SP:SAMPLEPREP_SUMMARY for 2 minutes at 4°C. The aqueous layer was removed to a new glass vial and SP:SAMPLEPREP_SUMMARY dried under N2 gas. The remaining organic layer in the bead beating tubes was SP:SAMPLEPREP_SUMMARY transferred into the glass centrifuge tube and the bead beating tube was rinsed SP:SAMPLEPREP_SUMMARY two more times with cold organic solvent. The combined organic rinses were SP:SAMPLEPREP_SUMMARY centrifuged, transferred to a new glass vial, and dried under N2 gas. Dried SP:SAMPLEPREP_SUMMARY aqueous fractions were re-dissolved in 380 µL of water. Dried organic fractions SP:SAMPLEPREP_SUMMARY were re-dissolved in 380 µL of 1:1 water:acetonitrile. 20 µL of SP:SAMPLEPREP_SUMMARY isotope-labeled injection standards in water were added to both fractions. SP:SAMPLEPREP_SUMMARY Process blanks (MilliQ water), media blanks, and PBS (vesicle suspension buffer) SP:SAMPLEPREP_SUMMARY were extracted and analyzed alongside each sample set. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Bligh-Dyer SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY See attached summary CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity UPLC HSS Cyano (100 x 2.1mm, 1.8um) CH:METHODS_FILENAME Ingalls_Lab_LC_Methods.txt #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Ingalls_Lab_MS_Methods.txt #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS See attached protocol. Data from organic fraction. MS:MS_RESULTS_FILE ST001524_AN002545_Results.txt UNITS:Adjusted and normalized peak areas Has m/z:Yes Has RT:Yes RT units:Minutes #END