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MB Sample ID: SA253778
| Local Sample ID: | 2243_Ac10L_3_TP6i |
| Subject ID: | SU002617 |
| Subject Type: | Bacteria |
| Subject Species: | Eggerthella lenta |
| Taxonomy ID: | 84112 |
| Genotype Strain: | DSM 2243, Valencia, AB8n2 |
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Subject:
| Subject ID: | SU002617 |
| Subject Type: | Bacteria |
| Subject Species: | Eggerthella lenta |
| Taxonomy ID: | 84112 |
| Genotype Strain: | DSM 2243, Valencia, AB8n2 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 2243_Ac10L_3_TP6i | SA253778 | FL032517 | 2243 | Strain |
| 2243_Ac10L_3_TP6i | SA253778 | FL032517 | 10L | AcetateGroup |
| 2243_Ac10L_3_TP6i | SA253778 | FL032517 | TP5 | TimePoint |
| 2243_Ac10L_3_TP6i | SA253778 | FL032517 | 39 | Time |
Collection:
| Collection ID: | CO002610 |
| Collection Summary: | Time course experiments were conducted in tubes in the anaerobic chamber in a 37°C incubator. For all metabolomics experiments, three independent culture replicates were included for each condition, with an equal number of uninoculated control tubes. Starter cultures and inocula were prepared as described above for growth assays. 5mLs of defined media was added to VWR glass culture tubes (53283-800) with screw caps. The PBS-washed inoculum was added to culture tubes to obtain an approximate starting OD600 of 0.001. A preliminary growth assay was conducted to define time points spanning the exponential growth phase in the tested conditions. At each time point, OD600 measurements of all inoculated tubes were first measured using a Hach DR1900 spectrophotometer, with a paired control tube to normalize for the background. 100 μL from each tube were then transferred into a 96-well microplate, which was sealed and removed from the anaerobic chamber. Plates were centrifuged at 1,928 rcf at 4°C for 8 minutes, after which supernatants were collected into fresh polypropylene tubes or plates, sealed, and flash-frozen in liquid nitrogen. Two time course experiments were carried out with stable isotope-labeled substrates. Experimental groups included conditions in which sodium acetate in the defined media was replaced with 13C2 labeled sodium acetate (Sigma-Aldrich 282014), along with a matched experimental group with the same concentration of unlabeled substrate. Intracellular extract samples were prepared with the following procedure, which was optimized for lysis of thick gram-positive cell walls: 600 μL of culture was transferred to an Eppendorf tube in anaerobic conditions and subsequently centrifuged at 10,000rcf for three minutes at 4°C, after which the supernatant was removed and the samples were immediately flash frozen to quench metabolites. 300 μL of cold methanol was then added to each pellet, followed by sonication on ice for 5 minutes and then shaking at 4°C for 4-12 hours. Samples were then centrifuged at 4°C at 15,000 rcf for 8 minutes, after which 120 μL of supernatant was transferred to fresh tubes and stored at -80°C until analysis. |
| Sample Type: | Bacterial culture supernatant |
| Collection Frequency: | at time points specified in study design table over 64 hours (full growth phase) |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002629 |
| Treatment Summary: | For growth and metabolomics experiments, glycerol stocks of the 3 E. lenta strains were first streaked on BHI+ agar plates and incubated at 37°C for 2-3 days. Individual colonies were inoculated into 3-4 mL liquid BHI+ and incubated at 37°C for 40-48 hours, or until approximately early stationary phase. Culture optical density (600 nm wavelength absorbance, OD600) was measured using a Hach DR1900 spectrophotometer. 1 mL samples of BHI starter cultures were then centrifuged at 1,568 rcf for 4 minutes in a microcentrifuge (ThermoScientific mySpin 12) in the anaerobic chamber and resuspended in 1 mL sterile phosphate-buffered saline (PBS). The resulting suspension was vortexed and diluted to an approximate OD600 of 0.1, and used as inoculum into defined experimental conditions. Varying media conditions were prepared separately and all allowed to fully reduce in the anaerobic chamber prior to inoculation. |
Sample Preparation:
| Sampleprep ID: | SP002623 |
| Sampleprep Summary: | Prior to analysis, intracellular samples were dried at room temperature via Centrivap Benchtop Concentrator (Labconco Corp.). Samples were re-suspended in 60 μL of a chilled solution of 1:1 methanol and acetonitrile, with 24% water at -20oC containing the internal standards CUDA and VAL-TYR-VAL each at 60 ng/mL. Samples were centrifuged at 4°C, 4,122 rcf for 5 minutes and the supernatant transferred to a vial and immediately capped for LC-MS analysis. |
Combined analysis:
| Analysis ID | AN004145 | AN004146 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | relative ion counts | relative ion counts |
Chromatography:
| Chromatography ID: | CH003069 |
| Chromatography Summary: | Samples, sterile media, pools, and blanks were promptly added to a Thermo Vanquish Autosampler at 4°C in a Vanquish UHPLC (Thermo Fisher Scientific, Waltham, MA). Chromatographic separation was performed using an ACQUITY Bridged Ethylene Hybrid (BEH) Amide column 2.1 x 150 mm, 1.7-micron particle size, (Waters Corp. Milford, MA), using chromatographic conditions published elsewhere (HILIC method described in the Supplementary Methods of doi.org/10.1038/s41586-021-03707-9). |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) |
| Column Temperature: | 40 |
| Flow Gradient: | The gradient profile was held at 100% B for 2 minutes, from 100% B to 70% B in 5 minutes, holding at 70% B for 0.7 minute, from 70% B to 40% B for 1.3 minutes, holding at 40% B for 0.5 minutes, from 40% B to 30% B for 0.75 minutes, before returning to 100% B for 2.5 minutes and holding at 100% B for 4 minutes. |
| Flow Rate: | 400 μL per minute |
| Solvent A: | 100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3 |
| Solvent B: | 95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003892 |
| Analysis ID: | AN004145 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. In SIRM samples, deuterated internal standards were replaced with CUDA and Val-Tyr-Val to enable untargeted enrichment analysis. LC-MS/MS analysis conditions for SIRM metabolomics were identical to those used for standard untargeted metabolomics. Intra- and extracellular untargeted data generated from SIRM experiments was analyzed separately using Compound Discoverer version 3.3 (Thermo Scientific, Bremen, Germany). Samples treated with labeled compounds were always paired with matched samples treated with unlabeled compounds in order to correct for naturally occurring isotope abundances. Unlabeled samples were used for compound detection and formula assignment via isotope pattern-based prediction, spectral library matches, or mass lists matches. The isotope patterns and formulas from the sample files then served as a reference for the detection of potential isotopologues per compound in the labeled sample type. A specification of the full Compound Discoverer workflow is available at https://github.com/turnbaughlab/2022_Noecker_ElentaMetabolism. |
| Ion Mode: | POSITIVE |
| MS ID: | MS003893 |
| Analysis ID: | AN004146 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. In SIRM samples, deuterated internal standards were replaced with CUDA and Val-Tyr-Val to enable untargeted enrichment analysis. LC-MS/MS analysis conditions for SIRM metabolomics were identical to those used for standard untargeted metabolomics. Intra- and extracellular untargeted data generated from SIRM experiments was analyzed separately using Compound Discoverer version 3.3 (Thermo Scientific, Bremen, Germany). Samples treated with labeled compounds were always paired with matched samples treated with unlabeled compounds in order to correct for naturally occurring isotope abundances. Unlabeled samples were used for compound detection and formula assignment via isotope pattern-based prediction, spectral library matches, or mass lists matches. The isotope patterns and formulas from the sample files then served as a reference for the detection of potential isotopologues per compound in the labeled sample type. A specification of the full Compound Discoverer workflow is available at https://github.com/turnbaughlab/2022_Noecker_ElentaMetabolism. |
| Ion Mode: | NEGATIVE |
