Summary of Study ST002509

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	ST002509
Study Title	Time course 1: Growth of Eggerthella lenta in defined media
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.
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	2023-03-17
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2023-04-04
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:	SU002609
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	Strain	AcetateConcentration_mM	Time
SA252438	2243_0_TP1_1_R2	2243	0	16.5
SA252439	2243_0_TP1_1_R3	2243	0	16.5
SA252440	2243_0_TP1_1_R1	2243	0	16.5
SA252441	2243_0_TP2_1_R3	2243	0	21.5
SA252442	2243_0_TP2_1_R2	2243	0	21.5
SA252443	2243_0_TP2_1_R1	2243	0	21.5
SA252444	2243_0_TP3_1_R3	2243	0	26
SA252445	2243_0_TP3_1_R2	2243	0	26
SA252446	2243_0_TP3_1_R1	2243	0	26
SA252447	2243_0_TP4_1_R3	2243	0	30
SA252448	2243_0_TP4_1_R2	2243	0	30
SA252449	2243_0_TP4_1_R1	2243	0	30
SA252450	2243_0_TP5_1_R3	2243	0	42
SA252451	2243_0_TP5_1_R2	2243	0	42
SA252452	2243_0_TP5_1_R1	2243	0	42
SA252453	2243_0_TP6_1_R3	2243	0	50
SA252454	2243_0_TP6_1_R2	2243	0	50
SA252455	2243_0_TP6_1_R1	2243	0	50
SA252456	2243_10Add_TP1_1_R3	2243	10Add	16.5
SA252457	2243_10Add_TP1_1_R2	2243	10Add	16.5
SA252458	2243_10Add_TP1_1_R1	2243	10Add	16.5
SA252459	2243_10Add_TP2_1_R3	2243	10Add	21.5
SA252460	2243_10Add_TP2_1_R2	2243	10Add	21.5
SA252461	2243_10Add_TP2_1_R1	2243	10Add	21.5
SA252462	2243_10Add_TP3_1_R1	2243	10Add	26
SA252463	2243_10Add_TP3_1_R3	2243	10Add	26
SA252464	2243_10Add_TP3_1_R2	2243	10Add	26
SA252465	2243_10Add_TP4_1_R1	2243	10Add	30
SA252466	2243_10Add_TP4_1_R2	2243	10Add	30
SA252467	2243_10Add_TP4_1_R3	2243	10Add	30
SA252468	2243_10Add_TP5_1_R2	2243	10Add	42
SA252469	2243_10Add_TP5_1_R3	2243	10Add	42
SA252470	2243_10Add_TP5_1_R1	2243	10Add	42
SA252471	2243_10Add_TP6_1_R3	2243	10Add	50
SA252472	2243_10Add_TP6_1_R2	2243	10Add	50
SA252473	2243_10Add_TP6_1_R1	2243	10Add	50
SA252474	2243_10_TP1_1_R3	2243	10	16.5
SA252475	2243_10_TP1_1_R1	2243	10	16.5
SA252476	2243_10_TP1_1_R2	2243	10	16.5
SA252477	2243_10_TP2_1_R2	2243	10	21.5
SA252478	2243_10_TP2_1_R1	2243	10	21.5
SA252479	2243_10_TP2_1_R3	2243	10	21.5
SA252480	2243_10_TP3_1_R2	2243	10	26
SA252481	2243_10_TP3_1_R3	2243	10	26
SA252482	2243_10_TP3_1_R1	2243	10	26
SA252483	2243_10_TP4_1_R3	2243	10	30
SA252484	2243_10_TP4_1_R1	2243	10	30
SA252485	2243_10_TP4_1_R2	2243	10	30
SA252486	2243_10_TP5_1_R2	2243	10	42
SA252487	2243_10_TP5_1_R1	2243	10	42
SA252488	2243_10_TP5_1_R3	2243	10	42
SA252489	2243_10_TP6_1_R3	2243	10	50
SA252490	2243_10_TP6_1_R1	2243	10	50
SA252491	2243_10_TP6_1_R2	2243	10	50
SA252492	2243_1_TP1_1_R2	2243	1	16.5
SA252493	2243_1_TP1_1_R1	2243	1	16.5
SA252494	2243_1_TP1_1_R3	2243	1	16.5
SA252495	2243_1_TP2_1_R1	2243	1	21.5
SA252496	2243_1_TP2_1_R2	2243	1	21.5
SA252497	2243_1_TP2_1_R3	2243	1	21.5
SA252498	2243_1_TP3_1_R2	2243	1	26
SA252499	2243_1_TP3_1_R3	2243	1	26
SA252500	2243_1_TP3_1_R1	2243	1	26
SA252501	2243_1_TP4_1_R1	2243	1	30
SA252502	2243_1_TP4_1_R2	2243	1	30
SA252503	2243_1_TP5_1_R2	2243	1	42
SA252504	2243_1_TP5_1_R1	2243	1	42
SA252505	2243_1_TP5_1_R3	2243	1	42
SA252506	2243_1_TP6_1_R2	2243	1	50
SA252507	2243_1_TP6_1_R3	2243	1	50
SA252508	2243_1_TP6_1_R1	2243	1	50
SA252509	AB8_0_TP1_1_R3	AB8	0	16.5
SA252510	AB8_0_TP1_1_R2	AB8	0	16.5
SA252511	AB8_0_TP1_1_R1	AB8	0	16.5
SA252512	AB8_0_TP2_1_R1	AB8	0	21.5
SA252513	AB8_0_TP2_1_R3	AB8	0	21.5
SA252514	AB8_0_TP2_1_R2	AB8	0	21.5
SA252515	AB8_0_TP3_1_R1	AB8	0	26
SA252516	AB8_0_TP3_1_R2	AB8	0	26
SA252517	AB8_0_TP4_1_R2	AB8	0	30
SA252518	AB8_0_TP4_1_R3	AB8	0	30
SA252519	AB8_0_TP4_1_R1	AB8	0	30
SA252520	AB8_0_TP5_1_R2	AB8	0	42
SA252521	AB8_0_TP5_1_R3	AB8	0	42
SA252522	AB8_0_TP5_1_R1	AB8	0	42
SA252523	AB8_0_TP6_1_R2	AB8	0	50
SA252524	AB8_0_TP6_1_R3	AB8	0	50
SA252525	AB8_0_TP6_1_R1	AB8	0	50
SA252526	AB8_10_TP1_1_R3	AB8	10	16.5
SA252527	AB8_10_TP1_1_R1	AB8	10	16.5
SA252528	AB8_10_TP1_1_R2	AB8	10	16.5
SA252529	AB8_10_TP2_1_R1	AB8	10	21.5
SA252530	AB8_10_TP2_1_R2	AB8	10	21.5
SA252531	AB8_10_TP2_1_R3	AB8	10	21.5
SA252532	AB8_10_TP3_1_R1	AB8	10	26
SA252533	AB8_10_TP3_1_R2	AB8	10	26
SA252534	AB8_10_TP3_1_R3	AB8	10	26
SA252535	AB8_10_TP4_1_R3	AB8	10	30
SA252536	AB8_10_TP4_1_R2	AB8	10	30
SA252537	AB8_10_TP4_1_R1	AB8	10	30

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Collection:

Collection ID:	CO002602
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.
Sample Type:	Bacterial culture supernatant
Collection Frequency:	at time points specified in study design table over 50 hours (full growth phase)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002621
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:

TR002621

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:	SP002615
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:

SP002615

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	AN004131	AN004132
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:	CH003061
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:	MS003878
Analysis ID:	AN004131
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. Metabolomics data was processed using MS-DIAL v4.60. Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average.
Ion Mode:	POSITIVE

MS ID:	MS003879
Analysis ID:	AN004132
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. Metabolomics data was processed using MS-DIAL v4.60. Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average.
Ion Mode:	NEGATIVE