Summary of Study ST002517

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	ST002517
Study Title	Time course 2: Growth of Eggerthella lenta in defined media with some samples receiving 13C2 stable isotope labeled acetate (intracellular samples)
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. Samples were collected at a subset of time points for extraction of intracellular metabolites.
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

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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:	SU002617
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	AcetateGroup	TimePoint	Time
SA253768	2243_Ac0_2_TP4i	2243	0	TP3	23
SA253769	2243_Ac0_3_TP4i	2243	0	TP3	23
SA253770	2243_Ac0_1_TP4i	2243	0	TP3	23
SA253771	2243_Ac0_3_TP6i	2243	0	TP5	39
SA253772	2243_Ac0_2_TP6i	2243	0	TP5	39
SA253773	2243_Ac0_1_TP6i	2243	0	TP5	39
SA253774	2243_Ac10L_3_TP4i	2243	10L	TP3	23
SA253775	2243_Ac10L_2_TP4i	2243	10L	TP3	23
SA253776	2243_Ac10L_1_TP4i	2243	10L	TP3	23
SA253777	2243_Ac10L_2_TP6i	2243	10L	TP5	39
SA253778	2243_Ac10L_3_TP6i	2243	10L	TP5	39
SA253779	2243_Ac10L_1_TP6i	2243	10L	TP5	39
SA253780	2243_Ac10_1_TP4i	2243	10	TP3	23
SA253781	2243_Ac10_2_TP4i	2243	10	TP3	23
SA253782	2243_Ac10_3_TP4i	2243	10	TP3	23
SA253783	2243_Ac10_3_TP6i	2243	10	TP5	39
SA253784	2243_Ac10_1_TP6i	2243	10	TP5	39
SA253785	2243_Ac10_2_TP6i	2243	10	TP5	39
SA253786	2243_Ac1L_1_TP4i	2243	1L	TP3	23
SA253787	2243_Ac1L_3_TP4i	2243	1L	TP3	23
SA253788	2243_Ac1L_2_TP4i	2243	1L	TP3	23
SA253789	2243_Ac1L_1_TP6i	2243	1L	TP5	39
SA253790	2243_Ac1L_2_TP6i	2243	1L	TP5	39
SA253791	2243_Ac1L_3_TP6i	2243	1L	TP5	39
SA253792	2243_Ac1_1_TP4i	2243	1	TP3	23
SA253793	2243_Ac1_2_TP4i	2243	1	TP3	23
SA253794	2243_Ac1_3_TP4i	2243	1	TP3	23
SA253795	2243_Ac1_3_TP6i	2243	1	TP5	39
SA253796	2243_Ac1_1_TP6i	2243	1	TP5	39
SA253797	2243_Ac1_2_TP6i	2243	1	TP5	39
SA253798	AB8n2_Ac0_1_TP4i	AB8n2	0	TP3	23
SA253799	AB8n2_Ac0_2_TP4i	AB8n2	0	TP3	23
SA253800	AB8n2_Ac0_3_TP4i	AB8n2	0	TP3	23
SA253801	AB8n2_Ac0_3_TP6i	AB8n2	0	TP5	39
SA253802	AB8n2_Ac0_1_TP6i	AB8n2	0	TP5	39
SA253803	AB8n2_Ac0_2_TP6i	AB8n2	0	TP5	39
SA253804	AB8n2_Ac10L_1_TP4i	AB8n2	10L	TP3	23
SA253805	AB8n2_Ac10L_3_TP4i	AB8n2	10L	TP3	23
SA253806	AB8n2_Ac10L_2_TP4i	AB8n2	10L	TP3	23
SA253807	AB8n2_Ac10L_2_TP6i	AB8n2	10L	TP5	39
SA253808	AB8n2_Ac10L_1_TP6i	AB8n2	10L	TP5	39
SA253809	AB8n2_Ac10L_3_TP6i	AB8n2	10L	TP5	39
SA253810	AB8n2_Ac10_1_TP4i	AB8n2	10	TP3	23
SA253811	AB8n2_Ac10_2_TP4i	AB8n2	10	TP3	23
SA253812	AB8n2_Ac10_3_TP4i	AB8n2	10	TP3	23
SA253813	AB8n2_Ac10_1_TP6i	AB8n2	10	TP5	39
SA253814	AB8n2_Ac10_2_TP6i	AB8n2	10	TP5	39
SA253815	AB8n2_Ac10_3_TP6i	AB8n2	10	TP5	39
SA253816	AB8n2_Ac1L_1_TP4i	AB8n2	1L	TP3	23
SA253817	AB8n2_Ac1L_3_TP4i	AB8n2	1L	TP3	23
SA253818	AB8n2_Ac1L_2_TP4i	AB8n2	1L	TP3	23
SA253819	AB8n2_Ac1L_1_TP6i	AB8n2	1L	TP5	39
SA253820	AB8n2_Ac1L_2_TP6i	AB8n2	1L	TP5	39
SA253821	AB8n2_Ac1L_3_TP6i	AB8n2	1L	TP5	39
SA253822	AB8n2_Ac1_1_TP4i	AB8n2	1	TP3	23
SA253823	AB8n2_Ac1_3_TP4i	AB8n2	1	TP3	23
SA253824	AB8n2_Ac1_2_TP4i	AB8n2	1	TP3	23
SA253825	AB8n2_Ac1_1_TP6i	AB8n2	1	TP5	39
SA253826	AB8n2_Ac1_2_TP6i	AB8n2	1	TP5	39
SA253827	AB8n2_Ac1_3_TP6i	AB8n2	1	TP5	39
SA253755	Intra_BK_6	-	-	-	-
SA253756	Intra_BK_7	-	-	-	-
SA253757	Intra_BK_5	-	-	-	-
SA253758	Intra_BK_4	-	-	-	-
SA253759	Intra_BK_3	-	-	-	-
SA253760	Intra_BK_8	-	-	-	-
SA253761	Intra_BK_9	-	-	-	-
SA253762	Intra_pool_4	-	-	-	-
SA253763	Intra_pool_3	-	-	-	-
SA253764	Intra_pool_2	-	-	-	-
SA253765	Intra_pool_1	-	-	-	-
SA253766	Intra_BK_1	-	-	-	-
SA253767	Intra_BK_2	-	-	-	-
SA253858	c_Ac0_2_TP4i	c	0	TP3	23
SA253859	c_Ac0_1_TP4i	c	0	TP3	23
SA253860	c_Ac0_2_TP6i	c	0	TP5	39
SA253861	c_Ac0_1_TP6i	c	0	TP5	39
SA253862	c_Ac10L_1_TP4i	c	10L	TP3	23
SA253863	c_Ac10L_2_TP4i	c	10L	TP3	23
SA253864	c_Ac10L_2_TP6i	c	10L	TP5	39
SA253865	c_Ac10L_1_TP6i	c	10L	TP5	39
SA253866	c_Ac10_2_TP4i	c	10	TP3	23
SA253867	c_Ac10_1_TP4i	c	10	TP3	23
SA253868	c_Ac10_1_TP6i	c	10	TP5	39
SA253869	c_Ac10_2_TP6i	c	10	TP5	39
SA253870	c_Ac1L_2_TP4i	c	1L	TP3	23
SA253871	c_Ac1L_1_TP4i	c	1L	TP3	23
SA253872	c_Ac1L_2_TP6i	c	1L	TP5	39
SA253873	c_Ac1L_1_TP6i	c	1L	TP5	39
SA253874	c_Ac1_1_TP4i	c	1	TP3	23
SA253875	c_Ac1_2_TP4i	c	1	TP3	23
SA253876	c_Ac1_1_TP6i	c	1	TP5	39
SA253877	c_Ac1_2_TP6i	c	1	TP5	39
SA253828	Valencia_Ac0_3_TP4i	Valencia	0	TP3	23
SA253829	Valencia_Ac0_1_TP4i	Valencia	0	TP3	23
SA253830	Valencia_Ac0_2_TP4i	Valencia	0	TP3	23
SA253831	Valencia_Ac0_1_TP6i	Valencia	0	TP5	39
SA253832	Valencia_Ac0_3_TP6i	Valencia	0	TP5	39
SA253833	Valencia_Ac0_2_TP6i	Valencia	0	TP5	39
SA253834	Valencia_Ac10L_3_TP4i	Valencia	10L	TP3	23

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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.

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.

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