Summary of Study ST001904
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 PR001199. The data can be accessed directly via it's Project DOI: 10.21228/M8QQ5J 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 | ST001904 |
| Study Title | Lipidomics analysis of outer membrane vesicles and elucidation of the ceramide phosphoinositol biosynthetic pathway in Bacteroides thetaiotaomicron |
| Study Type | Lipidic profile in wild-type and mutant strains |
| Study Summary | In this work, we characterized the lipid composition of membranes and OMV from Bacteroides thetaiotaomicron VPI-5482. LC-MS analysis indicate that OMV carry sphingolipids, glycerophospholipids and serine-dipeptide lipids. Sphingolipid species represent more than 50% of the total lipid content of OMV. The most abundant sphingolipids in OMV are ceramide phosphoethanolamine (CerPE) and ceramide phosphoinositol (CerPI). Bioinformatic analysis allowed the identification of the BT1522-1526 operon putatively involved in CerPI synthesis. Mutagenesis studies revealed BT1522-1526 are essential for synthesis of PI and CerPI, confirming the role of this operon in biosynthesis of CerPI. BT1522-1526 mutant strains lacking CerPI produced OMV that were indistinguishable from the wild-type strain, indicating that CerPI sphingolipid species are not involved in OMV biogenesis. Bacteroides sphingolipids are thought to modulate host-commensal interactions, and based on our data, we propose that OMV could act as long distance delivery vehicles for these molecules. |
| Institute | Washington University in St. Louis |
| Department | Molecular Microbiology |
| Laboratory | Feldman lab |
| Last Name | Sartorio |
| First Name | Mariana |
| Address | 660 S Euclid avenue, campus box 8230, 63110 |
| mgsartorio@wustl.edu | |
| Phone | 3147474477 |
| Submit Date | 2021-06-22 |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-08-30 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001199 |
| Project DOI: | doi: 10.21228/M8QQ5J |
| Project Title: | Lipidomic study |
| Project Type: | LC-MS analysis |
| Project Summary: | Lipidomic analysis of total membranes and outer membrane vesicles from the human gut comensal Bacteroides thetaiotaomicron. Strains used to perform the analysis: wild-tipe and mutants lacking the genes BT1522, BT1523, BT1524 and BT1526, involved in the synthesis of phosphoinosytol and Ceramide phosphinosytol |
| Institute: | Washington University in St. Louis |
| Department: | Molecular Microbiology |
| Laboratory: | Feldman lab |
| Last Name: | Sartorio |
| First Name: | Mariana |
| Address: | 660 S Euclid avenue, campus box 8230, 63110 |
| Email: | mgsartorio@wustl.edu |
| Phone: | 3147474477 |
| Funding Source: | NIH |
| Publications: | Lipidomics analysis of outer membrane vesicles and elucidation of the ceramide phosphoinositol biosynthetic pathway in Bacteroides thetaiotaomicron |
| Contributors: | Ezequiel Valguarnera, Mariana G. Sartorio, Fong-Fu Hsu and Mario F. Feldman |
Subject:
| Subject ID: | SU001982 |
| Subject Type: | Bacteria |
| Subject Species: | Bacteroides thetaiotaomicron |
| Taxonomy ID: | 818 |
Factors:
Subject type: Bacteria; Subject species: Bacteroides thetaiotaomicron (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA176592 | TM-1522-1 | deltaBT1522 |
| SA176593 | OMV-1522-2 | deltaBT1522 |
| SA176594 | OMV-1522-1 | deltaBT1522 |
| SA176595 | TM-1522-2 | deltaBT1522 |
| SA176596 | TM-1523-1 | deltaBT1523 |
| SA176597 | OMV-1523-1 | deltaBT1523 |
| SA176598 | TM-1523-2 | deltaBT1523 |
| SA176599 | OMV-1523-2 | deltaBT1523 |
| SA176600 | OMV-1524-1 | deltaBT1524 |
| SA176601 | OMV-1524-2 | deltaBT1524 |
| SA176602 | TM-1524-2 | deltaBT1524 |
| SA176603 | TM-1524-1 | deltaBT1524 |
| SA176604 | OMV-1526-1 | deltaBT1526 |
| SA176605 | OMV-1526-2 | deltaBT1526 |
| SA176606 | TM-1526-2 | deltaBT1526 |
| SA176607 | TM-1526-1 | deltaBT1526 |
| SA176608 | TM-WT-1 | wt |
| SA176609 | OMV-WT-2 | wt |
| SA176610 | OMV-WT-1 | wt |
| SA176611 | TM-WT-2 | wt |
| Showing results 1 to 20 of 20 |
Collection:
| Collection ID: | CO001975 |
| Collection Summary: | B. thetaiotaomicron strains (wild-type and ΔBT1522-BT1526 mutants) were grown overnight in an anaerobic chamber (Coy Laboratories) using an atmosphere of 10% H2, 5% CO2, 85% N2. For liquid growth, Brain heart infusion (BHI) supplemented with hemin and vitamin K3 was used. Cultures were then subjected to subcellular fractionation to obtain total membranes and outer membrane vesicles (OMV) preparation. OMV preparations: OMV were purified by ultracentrifugation of filtered spent media from 150 ml of liquid culture as described previously (1). OMV preparations were resuspended in PBS before lipid analyses. Protein content was quantified using a DC protein assay kit (Bio-Rad). Fractions were aliquoted and stored at -80°C until analyzed. Membrane preparations: Total membrane preparations were performed by cell lysis and ultracentrifugation as previously described (1). Total membranes from 150 ml of liquid culture were resuspended in PBS using a 2-ml glass tissue grinder with a polytetrafluoroethylene (PTFE) pestle (VWR). Protein content was quantified using a DC protein assay kit (Bio-Rad). Fractions were stored aliquoted and stored at -80°C until analyzed. References: 1. Elhenawy W, Debelyy MO, Feldman MF. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles. mBio. 2014;5(2):e00909-14. |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR001994 |
| Treatment Summary: | No treatment displayed. Wild-type and mutant bacteria were grown in BHI supplemented with vitamin K and Hemin |
Sample Preparation:
| Sampleprep ID: | SP001988 |
| Sampleprep Summary: | Total lipids from OMV and TM were extracted based on the Bligh and Dyer chloroform:methanol method (1). Briefly, 2 volumes of methanol, 1 volume of chloroform, and 0.8 volumes of Milli-Q water were added to 1 volume of PBS-resuspended OMV or TM fractions into solvent-resistant glass tubes. Contents were mixed for 1 min by vortexing and 1 volume of chloroform was added to the mixture. Contents were mixed for another minute and tubes were centrifuged for 5 min at 4000 rpm. After centrifugation, bottom phase (organic) was recovered using a glass Pasteur pipette and stored in solvent-sealed vials at -80°C until lipid analysis by LC-MS. References: 1. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37(8):911-7. |
Combined analysis:
| Analysis ID | AN003101 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Agilent 6550 |
| Column | Thermo Betasil C18 (100 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6550 QTOF |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002288 |
| Chromatography Summary: | LC/MS analyses were conducted on an Agilent 6550 A QTOF instrument with an Agilent 1290 HPLC with autosampler, operated by Agilent Masshunter software (Santa Clara, CA USA). Separation of the total lipid extracts was achieved by a Thermo (Waltham MA, USA) 100 × 2.1 mm BETASIL 5 μm C18 column at a flow rate of 300 μl/min at room temperature. The mobile phase contained 5 mM ammonium formate (pH 5.0) both in solvent A, acetonitrile:water (60:40, v/v), and solvent B, isopropanol:acetonitrile (90:10, v/v). A gradient elution in the following manner was applied: 68% A, 0–1.5 min; 68–55% A, 1.5–4 min; 55–48% A, 4–5 min; 48–42% A, 5–8 min; 42–34% A, 8–11 min; 34–30% A, 11–14 min; 30–25% A, 14–18 min; 25–3% A, 18–23 min; 3–0% A, 25–30 min; 0% A, 30–35 min; 68% A, 35–40 min. Both the positive-ion and negative-ion ESI MS scans were acquired in the mass range of 200-2000 Da at a rate of 2 scans/min. |
| Instrument Name: | Agilent 6550 |
| Column Name: | Thermo Betasil C18 (100 x 2.1mm,5um) |
| Column Temperature: | RT |
| Flow Gradient: | 68% A, 0-1.5 min; 68-55% A, 1.5-4 min; 55-48% A, 4-5 min; 48-42% A, 5-8 min; 42-34% A, 8-11 min; 34-30% A, 11-14 min; 30-25% A, 14-18 min; 25-3% A, 18-23 min; 3-0% A, 25-30 min; 0% A, 30-35 min; 68% A, 35-40 min |
| Flow Rate: | 300ul/min |
| Solvent A: | 60% acetonitrile/40% water; 5 mM ammonium formate, pH 5.0 |
| Solvent B: | 90% isopropanol/10% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002883 |
| Analysis ID: | AN003101 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | ESI MS scans were acquired in the mass range of 200-2000 Da at a rate of 2 scans /min. |
| Ion Mode: | UNSPECIFIED |
