Summary of Study ST001524
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001025. The data can be accessed directly via it's Project DOI: 10.21228/M86M5V This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001524 |
| Study Title | Prochlorococcus extracellular vesicles: Molecular composition and adsorption to diverse microbial cells |
| Study Type | Characterizing the metabolome of Prochlorococcus cells and vesicles |
| Study Summary | Extracellular vesicles are small (~50–200 nm diameter) membrane-bound structures released by cells from all domains of life. While extremely abundant in the oceans, our understanding of their functions, both for cells and the emergent ecosystem, is in its infancy. To advance this understanding, we analyzed the lipid, metabolite, and protein content of vesicles produced by two strains of the most abundant phytoplankton cell in the ocean, the cyanobacterium Prochlorococcus. We show that Prochlorococcus exports an enormous array of cellular compounds into their surroundings via extracellular vesicles. The vesicles produced by the two different strains contained some materials in common, but also displayed numerous strain-specific differences, reflecting functional complexity within natural vesicle populations. Prochlorococcus vesicles contain active enzymes, indicating that they can mediate biogeochemically relevant extracellular reactions in the wild. Interaction assays demonstrate that vesicles from Prochlorococcus and multiple genera of heterotrophic bacteria can associate with other marine microbes, including Pelagibacter, the most abundant heterotrophic group in the oceans. Our observations suggest that vesicles may play diverse functional roles in the oceans, including but not limited to mediating energy and nutrient transfers, catalyzing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species. These findings further indicate that a portion of the ‘dissolved’ compounds in the oceans are not truly dissolved, but are instead packaged within locally structured, colloidal vesicles. |
| Institute | University of Washington |
| Department | Oceanography |
| Laboratory | Ingalls Lab |
| Last Name | Carlson |
| First Name | Laura |
| Address | 1501 NE Boat Street, Marine Science Building, Room G, Seattle, WA 98195 |
| truxal@uw.edu | |
| Phone | 4125545093 |
| Submit Date | 2020-11-04 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-05-04 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001025 |
| Project DOI: | doi: 10.21228/M86M5V |
| Project Title: | Prochlorococcus extracellular vesicles: Molecular composition and adsorption to diverse microbial cells |
| Project Type: | Marine Metabolomics |
| Project Summary: | Extracellular vesicles are small (~50–200 nm diameter) membrane-bound structures released by cells from all domains of life. While extremely abundant in the oceans, our understanding of their functions, both for cells and the emergent ecosystem, is in its infancy. To advance this understanding, we analyzed the lipid, metabolite, and protein content of vesicles produced by two strains of the most abundant phytoplankton cell in the ocean, the cyanobacterium Prochlorococcus. We show that Prochlorococcus exports an enormous array of cellular compounds into their surroundings via extracellular vesicles. The vesicles produced by the two different strains contained some materials in common, but also displayed numerous strain-specific differences, reflecting functional complexity within natural vesicle populations. Prochlorococcus vesicles contain active enzymes, indicating that they can mediate biogeochemically relevant extracellular reactions in the wild. Interaction assays demonstrate that vesicles from Prochlorococcus and multiple genera of heterotrophic bacteria can associate with other marine microbes, including Pelagibacter, the most abundant heterotrophic group in the oceans. Our observations suggest that vesicles may play diverse functional roles in the oceans, including but not limited to mediating energy and nutrient transfers, catalyzing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species. These findings further indicate that a portion of the ‘dissolved’ compounds in the oceans are not truly dissolved, but are instead packaged within locally structured, colloidal vesicles. |
| Institute: | University of Washington |
| Department: | Oceanography |
| Laboratory: | Ingalls Lab |
| Last Name: | Carlson |
| First Name: | Laura |
| Address: | 1501 NE Boat Street, Marine Science Building, Room G, Seattle, WA 98195 |
| Email: | truxal@uw.edu |
| Phone: | 4125545093 |
Subject:
| Subject ID: | SU001598 |
| Subject Type: | Other |
| Subject Species: | Prochlorococcus marinus str. MIT 9312;Prochlorococcus marinus MIT9313 |
| Taxonomy ID: | 74546;74547 |
Factors:
Subject type: Other; Subject species: Prochlorococcus marinus str. MIT 9312;Prochlorococcus marinus MIT9313 (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample Type | Cell Type |
|---|---|---|---|
| SA128430 | Cell_9312_A | Pellet | 9312 |
| SA128431 | Cell_9312_C | Pellet | 9312 |
| SA128432 | Cell_9312_B | Pellet | 9312 |
| SA128433 | Cell_9313_C | Pellet | 9313 |
| SA128434 | Cell_9313_A | Pellet | 9313 |
| SA128435 | Cell_9313_B | Pellet | 9313 |
| SA128436 | Vesicle_9312_3 | Vesicle | 9312 |
| SA128437 | Vesicle_9312_1 | Vesicle | 9312 |
| SA128438 | Vesicle_9312_2 | Vesicle | 9312 |
| SA128439 | Vesicle_9313_3 | Vesicle | 9313 |
| SA128440 | Vesicle_9313_2 | Vesicle | 9313 |
| SA128441 | Vesicle_9313_1 | Vesicle | 9313 |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO001593 |
| Collection Summary: | Axenic cultures of Prochlorococcus strain MIT9312 and MIT9313 were grown in defined artificial AMP1 media supplemented with 10 mM (final concentration) filter-sterilized sodium bicarbonate. Seven 20 L cultures were grown for each of the two Prochlorococcus strains, providing three replicates for the lipid and small metabolite analysis and an additional sample for proteomics analysis. Strains MIT9312 and MIT9313 are available from the National Center for Marine Algae and Microbiota. 20 L cultures were grown in polycarbonate carboys (ThermoFisher Nalgene, Waltham, MA, USA) with gentle stirring (60 rpm), under constant light flux (10 – 20 µmol Q m -2 s -1 for MIT9313; 30 – 40 µmol Q m -2 s -1 for MIT9312) at 24°C. Vesicles were collected from 20 L cultures of Prochlorococcus during mid-to-late exponential growth phase and isolated as described previously (Biller et al., 2014, Science). Briefly, cultures were first gravity filtered through a 0.2 µm capsule filter (Polycap 150TC; GE Life Sciences/Whatman, Maidstone, UK). The filtrate was then concentrated using a 100 kDa tangential flow filter (Ultrasette with Omega membrane; Pall, Port Washington, NY, USA) and re-filtered through a 0.2 µm syringe filter. Vesicles were pelleted from the sample by ultracentrifugation at ~100,000 x g (Beckman-coulter SW32Ti rotor; 32,000 rpm, 1.5 hrs, 4°C), purified on an OptiPrep gradient (Biller et al., 2014, Science), then washed and resuspended in 0.2 µm filtered 1x PBS. |
| Sample Type: | Cultured Prochlorococcus cells and vesicles |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001613 |
| Treatment Summary: | No treatment - cells and vesicles were cultured according to standard protocols. We used targeted and untargeted metabolomics to characterize the metabolome of cells and vesicles. |
Sample Preparation:
| Sampleprep ID: | SP001606 |
| Sampleprep Summary: | Each sample was extracted using a modified Bligh-Dyer extraction. Briefly, quantitative aliquots of cell pellets were transferred into 15 mL teflon centrifuge tubes containing a mixture of 100 µm and 400 µm silica beads. Quantitative aliquots of extracellular vesicles were transferred into 24 mL glass vials and extracted without bead beating. Heavy isotope-labeled internal standards were added along with ~2 mL of cold aqueous solvent (50:50 methanol:water) and ~3 mL of cold organic solvent (dichloromethane). The samples were shaken on a FastPrep-24 Homogenizer for 30 seconds and chilled in a -20 °C freezer repeatedly for three cycles of bead-beating and a total of 30 minutes of chilling. The organic and aqueous layers were separated by spinning samples in a centrifuge at 4,300 rpm for 2 minutes at 4 °C. The aqueous layer was removed to a new glass centrifuge tube. The remaining organic fraction was rinsed three more times with additions of 1 to 2 mL of 50:50 methanol:water. All aqueous rinses were combined for each sample and ~2 mL of cold dichloromethane was added to the combined aqueous layer. Tubes were shaken and centrifuged at 4,300 rpm for 2 minutes at 4°C. The aqueous layer was removed to a new glass vial and dried under N2 gas. The remaining organic layer in the bead beating tubes was transferred into the glass centrifuge tube and the bead beating tube was rinsed two more times with cold organic solvent. The combined organic rinses were centrifuged, transferred to a new glass vial, and dried under N2 gas. Dried aqueous fractions were re-dissolved in 380 µL of water. Dried organic fractions were re-dissolved in 380 µL of 1:1 water:acetonitrile. 20 µL of isotope-labeled injection standards in water were added to both fractions. Process blanks (MilliQ water), media blanks, and PBS (vesicle suspension buffer) were extracted and analyzed alongside each sample set. |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Bligh-Dyer |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN002542 | AN002543 | AN002544 | AN002545 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | HILIC | HILIC | Reversed phase | Reversed phase |
| Chromatography system | Waters Acquity I-Class | Waters Acquity I-Class | Waters Acquity I-Class | Waters Acquity I-Class |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) | Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | POSITIVE |
| Units | Adjusted and normalized peak areas | Adjusted and normalized peak areas | Adjusted and normalized peak areas | Adjusted and normalized peak areas |
Chromatography:
| Chromatography ID: | CH001862 |
| Chromatography Summary: | See attached summary. |
| Methods Filename: | Ingalls_Lab_LC_Methods.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 30 |
| Flow Rate: | 0.15 ml/min |
| Solvent A: | 85% acetonitrile/15% water; 10 mM ammonium carbonate |
| Solvent B: | 15% acetonitrile/85% water; 10 mM ammonium carbonate |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH001863 |
| Chromatography Summary: | See attached summary |
| Methods Filename: | Ingalls_Lab_LC_Methods.txt |
| Instrument Name: | Waters Acquity I-Class |
| Column Name: | Waters Acquity UPLC HSS Cyano (100 x 2.1mm,1.8um) |
| Column Temperature: | 35 |
| Flow Rate: | 0.4 ml/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002360 |
| Analysis ID: | AN002542 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Ingalls_Lab_MS_Methods.txt |
| MS ID: | MS002361 |
| Analysis ID: | AN002543 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | Ingalls_Lab_MS_Methods.txt |
| MS ID: | MS002362 |
| Analysis ID: | AN002544 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. Data from aqueous fraction. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Ingalls_Lab_MS_Methods.txt |
| MS ID: | MS002363 |
| Analysis ID: | AN002545 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See attached protocol. Data from organic fraction. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Ingalls_Lab_MS_Methods.txt |
