Summary of Study ST002516

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 PR001620. The data can be accessed directly via it's Project DOI: 10.21228/M89B04 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	ST002516
Study Title	Time course 2: Growth of Eggerthella lenta in defined media with some samples receiving 13C2 stable isotope labeled acetate
Study Type	Untargeted LC-MS
Study Summary	This dataset contains untargeted metabolomics analysis of supernatants from 3 strains of Eggerthella lenta grown in defined EDM1 media with varying acetate concentrations. One set of samples grew in EDM1 containing 13C2 stable isotope labeled acetate.
Institute	University of California, San Francisco
Last Name	Noecker
First Name	Cecilia
Address	513 Parnassus Ave HSW1501, San Francisco, CA 94143
Email	cecilia.noecker@ucsf.edu
Phone	415-502-3264
Submit Date	2023-03-22
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2023-04-10
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001620
Project DOI:	doi: 10.21228/M89B04
Project Title:	Systems biology illuminates the alternative metabolic niche of the human gut bacterium Eggerthella lenta
Project Type:	Untargeted LC-MS
Project Summary:	Human gut bacteria perform diverse metabolic functions with consequences for host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta performs several unusual chemical transformations, but it does not metabolize sugars and its core growth strategy remains unclear. To obtain a comprehensive view of the metabolic network of E. lenta, we generated several complementary resources: defined culture media, metabolomics profiles of strain isolates, and a curated genome-scale metabolic reconstruction. Stable isotope-resolved metabolomics revealed that E. lenta uses acetate as a key carbon source while catabolizing arginine to generate ATP, traits which could be recapitulated in silico by our updated metabolic model. We compared these in vitro findings with metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying shared signatures across environments and highlighting catabolism of the host signaling metabolite agmatine as an alternative energy pathway. Together, our results elucidate a distinctive metabolic niche filled by E. lenta in the gut ecosystem.
Institute:	University of California, San Francisco
Department:	Microbiology and Immunology
Laboratory:	Peter Turnbaugh
Last Name:	Noecker
First Name:	Cecilia
Address:	513 Parnassus Ave HSW1501, San Francisco, CA 94143
Email:	cecilia.noecker@ucsf.edu
Phone:	415-502-3264
Funding Source:	This work was supported by the National Institutes of Health (2R01HL122593; 1R01AT011117; 1R01DK114034 to P.J.T., F32GM140808 to C.N.). P.J.T. is a Chan Zuckerberg Biohub Investigator and held an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.
Publications:	https://doi.org/10.1101/2022.09.19.508335

Subject:

Subject ID:	SU002616
Subject Type:	Bacteria
Subject Species:	Eggerthella lenta
Taxonomy ID:	84112
Genotype Strain:	DSM 2243, Valencia, AB8n2

Factors:

Subject type: Bacteria; Subject species: Eggerthella lenta (Factor headings shown in green)

mb_sample_id	local_sample_id	Strain2	AcetateGroup	TimePoint	Time
SA253216	2243_0_TP0_1	2243	0	TP0	0
SA253217	2243_0_TP0_3	2243	0	TP0	0
SA253218	2243_0_TP0_2	2243	0	TP0	0
SA253219	2243_0_TP1_3	2243	0	TP1	15
SA253220	2243_0_TP1_1	2243	0	TP1	15
SA253221	2243_0_TP1_2	2243	0	TP1	15
SA253222	2243_0_TP2_2	2243	0	TP2	18.5
SA253223	2243_0_TP2_3	2243	0	TP2	18.5
SA253224	2243_0_TP2_1	2243	0	TP2	18.5
SA253225	2243_0_TP3_3	2243	0	TP3	23
SA253226	2243_0_TP3_2	2243	0	TP3	23
SA253227	2243_0_TP3_1	2243	0	TP3	23
SA253228	2243_0_TP4_3	2243	0	TP4	28.5
SA253229	2243_0_TP4_1	2243	0	TP4	28.5
SA253230	2243_0_TP4_2	2243	0	TP4	28.5
SA253231	2243_0_TP5_3	2243	0	TP5	39
SA253232	2243_0_TP5_1	2243	0	TP5	39
SA253233	2243_0_TP5_2	2243	0	TP5	39
SA253234	2243_0_TP6_1	2243	0	TP6	43
SA253235	2243_0_TP6_3	2243	0	TP6	43
SA253236	2243_0_TP6_2	2243	0	TP6	43
SA253237	2243_0_TP7_3	2243	0	TP7	47.5
SA253238	2243_0_TP7_2	2243	0	TP7	47.5
SA253239	2243_0_TP7_1	2243	0	TP7	47.5
SA253240	2243_0_TP8_1	2243	0	TP8	64
SA253241	2243_0_TP8_2	2243	0	TP8	64
SA253242	2243_0_TP8_3	2243	0	TP8	64
SA253243	2243_10L_TP0_1	2243	10L	TP0	0
SA253244	2243_10L_TP0_3	2243	10L	TP0	0
SA253245	2243_10L_TP0_2	2243	10L	TP0	0
SA253246	2243_10L_TP1_3	2243	10L	TP1	15
SA253247	2243_10L_TP1_1	2243	10L	TP1	15
SA253248	2243_10L_TP1_2	2243	10L	TP1	15
SA253249	2243_10L_TP2_1	2243	10L	TP2	18.5
SA253250	2243_10L_TP2_2	2243	10L	TP2	18.5
SA253251	2243_10L_TP2_3	2243	10L	TP2	18.5
SA253252	2243_10L_TP3_1	2243	10L	TP3	23
SA253253	2243_10L_TP3_2	2243	10L	TP3	23
SA253254	2243_10L_TP3_3	2243	10L	TP3	23
SA253255	2243_1L_TP4_2	2243	10L	TP4	28.5
SA253256	2243_1L_TP4_1	2243	10L	TP4	28.5
SA253257	2243_1L_TP4_3	2243	10L	TP4	28.5
SA253258	2243_1L_TP5_3	2243	10L	TP5	39
SA253259	2243_1L_TP5_1	2243	10L	TP5	39
SA253260	2243_1L_TP5_2	2243	10L	TP5	39
SA253261	2243_1L_TP6_3	2243	10L	TP6	43
SA253262	2243_1L_TP6_2	2243	10L	TP6	43
SA253263	2243_1L_TP6_1	2243	10L	TP6	43
SA253264	2243_1L_TP7_2	2243	10L	TP7	47.5
SA253265	2243_1L_TP7_1	2243	10L	TP7	47.5
SA253266	2243_1L_TP7_3	2243	10L	TP7	47.5
SA253267	2243_10L_TP8_1	2243	10L	TP8	64
SA253268	2243_10L_TP8_3	2243	10L	TP8	64
SA253269	2243_10L_TP8_2	2243	10L	TP8	64
SA253270	2243_10_TP0_2	2243	10	TP0	0
SA253271	2243_10_TP0_3	2243	10	TP0	0
SA253272	2243_10_TP0_1	2243	10	TP0	0
SA253273	2243_10_TP1_2	2243	10	TP1	15
SA253274	2243_10_TP1_1	2243	10	TP1	15
SA253275	2243_10_TP1_3	2243	10	TP1	15
SA253276	2243_10_TP2_2	2243	10	TP2	18.5
SA253277	2243_10_TP2_3	2243	10	TP2	18.5
SA253278	2243_10_TP2_1	2243	10	TP2	18.5
SA253279	2243_10_TP3_1	2243	10	TP3	23
SA253280	2243_10_TP3_3	2243	10	TP3	23
SA253281	2243_10_TP3_2	2243	10	TP3	23
SA253282	2243_1_TP4_3	2243	10	TP4	28.5
SA253283	2243_1_TP4_2	2243	10	TP4	28.5
SA253284	2243_1_TP4_1	2243	10	TP4	28.5
SA253285	2243_1_TP5_2	2243	10	TP5	39
SA253286	2243_1_TP5_1	2243	10	TP5	39
SA253287	2243_1_TP5_3	2243	10	TP5	39
SA253288	2243_1_TP6_1	2243	10	TP6	43
SA253289	2243_1_TP6_3	2243	10	TP6	43
SA253290	2243_1_TP6_2	2243	10	TP6	43
SA253291	2243_1_TP7_2	2243	10	TP7	47.5
SA253292	2243_1_TP7_3	2243	10	TP7	47.5
SA253293	2243_1_TP7_1	2243	10	TP7	47.5
SA253294	2243_10_TP8_3	2243	10	TP8	64
SA253295	2243_10_TP8_2	2243	10	TP8	64
SA253296	2243_10_TP8_1	2243	10	TP8	64
SA253297	2243_1L_TP0_1	2243	1L	TP0	0
SA253298	2243_1L_TP0_3	2243	1L	TP0	0
SA253299	2243_1L_TP0_2	2243	1L	TP0	0
SA253300	2243_1L_TP1_2	2243	1L	TP1	15
SA253301	2243_1L_TP1_1	2243	1L	TP1	15
SA253302	2243_1L_TP1_3	2243	1L	TP1	15
SA253303	2243_1L_TP2_1	2243	1L	TP2	18.5
SA253304	2243_1L_TP2_2	2243	1L	TP2	18.5
SA253305	2243_1L_TP2_3	2243	1L	TP2	18.5
SA253306	2243_1L_TP3_3	2243	1L	TP3	23
SA253307	2243_1L_TP3_2	2243	1L	TP3	23
SA253308	2243_1L_TP3_1	2243	1L	TP3	23
SA253309	2243_10L_TP4_2	2243	1L	TP4	28.5
SA253310	2243_10L_TP4_1	2243	1L	TP4	28.5
SA253311	2243_10L_TP4_3	2243	1L	TP4	28.5
SA253312	2243_10L_TP5_1	2243	1L	TP5	39
SA253313	2243_10L_TP5_2	2243	1L	TP5	39
SA253314	2243_10L_TP6_3	2243	1L	TP6	43
SA253315	2243_10L_TP6_2	2243	1L	TP6	43

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Collection:

Collection ID:	CO002609
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.
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:	TR002628
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.

Treatment ID:

TR002628

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:	SP002622
Sampleprep Summary:	Bacterial culture supernatant and sterile media, used in culture, were thawed on wet ice. Once thawed, samples were homogenized by inversion five times. Extracellular culture supernatant samples were prepared as follows: 20 μL of culture supernatant were extracted using 80 μL of a chilled extraction solvent at −20°C (1:1 acetonitrile:methanol, 5% water containing stable isotope-labeled internal standards). Samples were homogenized via pipette action, incubated for 1 hour at −20°C, centrifuged at 4°C at 6000 rcf for 5 min. The supernatant was transferred to a new plate and immediately sealed and kept at 4°C prior to prompt analysis via LC-MS/MS.

Sampleprep ID:

SP002622

Sampleprep Summary:

Bacterial culture supernatant and sterile media, used in culture, were thawed on wet ice. Once thawed, samples were homogenized by inversion five times. Extracellular culture supernatant samples were prepared as follows: 20 μL of culture supernatant were extracted using 80 μL of a chilled extraction solvent at −20°C (1:1 acetonitrile:methanol, 5% water containing stable isotope-labeled internal standards). Samples were homogenized via pipette action, incubated for 1 hour at −20°C, centrifuged at 4°C at 6000 rcf for 5 min. The supernatant was transferred to a new plate and immediately sealed and kept at 4°C prior to prompt analysis via LC-MS/MS.

Combined analysis:

Analysis ID	AN004143	AN004144
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:	CH003068
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:	MS003890
Analysis ID:	AN004143
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:	MS003891
Analysis ID:	AN004144
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