Summary of Study ST002510

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	ST002510
Study Title	Strain supernatants: Strain diversity of Eggerthella lenta metabolites in defined media
Study Type	Untargeted LC-MS
Study Summary	This dataset contains untargeted metabolomics analysis of supernatants from stationary phase of a collection of 30 strains of Eggerthella lenta grown in defined EDM1 media.
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

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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:	SU002610
Subject Type:	Bacteria
Subject Species:	Eggerthella lenta
Taxonomy ID:	84112
Genotype Strain:	various (see sample table)

Factors:

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

mb_sample_id	local_sample_id	Strain	Strain name	Condition	PlateNum
SA252743	Ctrl_B3_1	Ctrl	Ctrl_B3	GL_Ctrl_B3	1
SA252744	Ctrl_B3_2	Ctrl	Ctrl_B3	GL_Ctrl_B3	2
SA252745	Ctrl_B3_3	Ctrl	Ctrl_B3	GL_Ctrl_B3	3
SA252746	Ctrl_D9_2	Ctrl	Ctrl_D9	GL_Ctrl_D9	2
SA252747	Ctrl_F5_1	Ctrl	Ctrl_F5	GL_Ctrl_F5	1
SA252748	Ctrl_F5_2	Ctrl	Ctrl_F5	GL_Ctrl_F5	2
SA252749	Ctrl_F5_3	Ctrl	Ctrl_F5	GL_Ctrl_F5	3
SA252750	Ctrl_G12_1	Ctrl	Ctrl_G12	GL_Ctrl_G12	1
SA252751	Ctrl_G12_2	Ctrl	Ctrl_G12	GL_Ctrl_G12	2
SA252752	Ctrl_G12_3	Ctrl	Ctrl_G12	GL_Ctrl_G12	3
SA252753	PTJ0086_1	PTJ0086	Eggerthella_lenta_1160AFAABroad	GL	1
SA252754	PTJ0086_2	PTJ0086	Eggerthella_lenta_1160AFAABroad	GL	2
SA252755	PTJ0086_3	PTJ0086	Eggerthella_lenta_1160AFAABroad	GL	3
SA252756	PTJ0087_1	PTJ0087	Eggerthella_lenta_1356FAA	GL	1
SA252757	PTJ0087_2	PTJ0087	Eggerthella_lenta_1356FAA	GL	2
SA252758	PTJ0087_3	PTJ0087	Eggerthella_lenta_1356FAA	GL	3
SA252759	PTJ0089_1	PTJ0089	Eggerthella_lenta_11C	GL	1
SA252760	PTJ0089_3	PTJ0089	Eggerthella_lenta_11C	GL	3
SA252761	PTJ0090_1	PTJ0090	Eggerthella_lenta_14A	GL	1
SA252762	PTJ0090_3	PTJ0090	Eggerthella_lenta_14A	GL	3
SA252763	PTJ0092_1	PTJ0092	Eggerthella_lenta_22C	GL	1
SA252764	PTJ0092_2	PTJ0092	Eggerthella_lenta_22C	GL	2
SA252765	PTJ0092_3	PTJ0092	Eggerthella_lenta_22C	GL	3
SA252766	PTJ0093_1	PTJ0093	Eggerthella_lenta_28B	GL	1
SA252767	PTJ0093_2	PTJ0093	Eggerthella_lenta_28B	GL	2
SA252768	PTJ0093_3	PTJ0093	Eggerthella_lenta_28B	GL	3
SA252769	PTJ0109_1	PTJ0109	Eggerthella_lenta_DSM11767	GL	1
SA252770	PTJ0109_2	PTJ0109	Eggerthella_lenta_DSM11767	GL	2
SA252771	PTJ0109_3	PTJ0109	Eggerthella_lenta_DSM11767	GL	3
SA252772	PTJ0110_1	PTJ0110	Eggerthella_lenta_DSM11863	GL	1
SA252773	PTJ0110_2	PTJ0110	Eggerthella_lenta_DSM11863	GL	2
SA252774	PTJ0110_3	PTJ0110	Eggerthella_lenta_DSM11863	GL	3
SA252775	PTJ0111_1	PTJ0111	Eggerthella_lenta_DSM15644	GL	1
SA252776	PTJ0111_2	PTJ0111	Eggerthella_lenta_DSM15644	GL	2
SA252777	PTJ0111_3	PTJ0111	Eggerthella_lenta_DSM15644	GL	3
SA252778	PTJ0112_1	PTJ0112	Eggerthella_sinensis_DSM16107	GL	1
SA252779	PTJ0112_2	PTJ0112	Eggerthella_sinensis_DSM16107	GL	2
SA252780	PTJ0112_3	PTJ0112	Eggerthella_sinensis_DSM16107	GL	3
SA252781	PTJ0166_1	PTJ0166	Eggerthella_lenta_UCSF2243	GL	1
SA252782	PTJ0166_2	PTJ0166	Eggerthella_lenta_UCSF2243	GL	2
SA252783	PTJ0166_3	PTJ0166	Eggerthella_lenta_UCSF2243	GL	3
SA252784	PTJ0169_1	PTJ0169	Eggerthella_lenta_Valencia	GL	1
SA252785	PTJ0169_2	PTJ0169	Eggerthella_lenta_Valencia	GL	2
SA252786	PTJ0169_3	PTJ0169	Eggerthella_lenta_Valencia	GL	3
SA252787	PTJ0170_1	PTJ0170	Eggerthella_lenta_AN51LG	GL	1
SA252788	PTJ0170_2	PTJ0170	Eggerthella_lenta_AN51LG	GL	2
SA252789	PTJ0170_3	PTJ0170	Eggerthella_lenta_AN51LG	GL	3
SA252790	PTJ0171_1	PTJ0171	Eggerthella_lenta_MR1n12	GL	1
SA252791	PTJ0171_2	PTJ0171	Eggerthella_lenta_MR1n12	GL	2
SA252792	PTJ0171_3	PTJ0171	Eggerthella_lenta_MR1n12	GL	3
SA252793	PTJ0173_2	PTJ0173	Eggerthella_lenta_326I6NA	GL	2
SA252794	PTJ0173_3	PTJ0173	Eggerthella_lenta_326I6NA	GL	3
SA252795	PTJ0174_1	PTJ0174	Eggerthella_lenta_AB8n2	GL	1
SA252796	PTJ0174_3	PTJ0174	Eggerthella_lenta_AB8n2	GL	3
SA252797	PTJ0175_1	PTJ0175	Eggerthella_lenta_AB12n2	GL	1
SA252798	PTJ0175_2	PTJ0175	Eggerthella_lenta_AB12n2	GL	2
SA252799	PTJ0175_3	PTJ0175	Eggerthella_lenta_AB12n2	GL	3
SA252800	PTJ0176_1	PTJ0176	Eggerthella_lenta_CC82BHI2	GL	1
SA252801	PTJ0176_2	PTJ0176	Eggerthella_lenta_CC82BHI2	GL	2
SA252802	PTJ0176_3	PTJ0176	Eggerthella_lenta_CC82BHI2	GL	3
SA252803	PTJ0177_1	PTJ0177	Eggerthella_lenta_RC46F	GL	1
SA252804	PTJ0177_2	PTJ0177	Eggerthella_lenta_RC46F	GL	2
SA252805	PTJ0177_3	PTJ0177	Eggerthella_lenta_RC46F	GL	3
SA252806	PTJ0178_1	PTJ0178	Eggerthella_lenta_CC75D52	GL	1
SA252807	PTJ0178_2	PTJ0178	Eggerthella_lenta_CC75D52	GL	2
SA252808	PTJ0178_3	PTJ0178	Eggerthella_lenta_CC75D52	GL	3
SA252809	PTJ0179_1	PTJ0179	Eggerthella_lenta_CC86D54	GL	1
SA252810	PTJ0179_2	PTJ0179	Eggerthella_lenta_CC86D54	GL	2
SA252811	PTJ0179_3	PTJ0179	Eggerthella_lenta_CC86D54	GL	3
SA252812	PTJ0180_1	PTJ0180	Eggerthella_lenta_W1BHI6	GL	1
SA252813	PTJ0180_2	PTJ0180	Eggerthella_lenta_W1BHI6	GL	2
SA252814	PTJ0180_3	PTJ0180	Eggerthella_lenta_W1BHI6	GL	3
SA252815	PTJ0237_1	PTJ0237	Eggerthella_lenta_A2	GL	1
SA252816	PTJ0237_2	PTJ0237	Eggerthella_lenta_A2	GL	2
SA252817	PTJ0237_3	PTJ0237	Eggerthella_lenta_A2	GL	3
SA252818	PTJ0240_1	PTJ0240	Eggerthella_lenta_ATCC25559	GL	1
SA252819	PTJ0240_2	PTJ0240	Eggerthella_lenta_ATCC25559	GL	2
SA252820	PTJ0240_3	PTJ0240	Eggerthella_lenta_ATCC25559	GL	3
SA252821	PTJ0261_1	PTJ0261	Eggerthella_lenta_DSM2243D	GL	1
SA252822	PTJ0261_2	PTJ0261	Eggerthella_lenta_DSM2243D	GL	2
SA252823	PTJ0261_3	PTJ0261	Eggerthella_lenta_DSM2243D	GL	3
SA252824	PTJ0289_1	PTJ0289	Eggerthella_lenta_ELUC2	GL	1
SA252825	PTJ0289_2	PTJ0289	Eggerthella_lenta_ELUC2	GL	2
SA252826	PTJ0289_3	PTJ0289	Eggerthella_lenta_ELUC2	GL	3
SA252827	PTJ0290_1	PTJ0290	Eggerthella_lenta_ELUC5	GL	1
SA252828	PTJ0290_2	PTJ0290	Eggerthella_lenta_ELUC5	GL	2
SA252829	PTJ0290_3	PTJ0290	Eggerthella_lenta_ELUC5	GL	3
SA252830	PTJ0295_1	PTJ0295	Eggerthella_lenta_RJX1627	GL	1
SA252831	PTJ0295_2	PTJ0295	Eggerthella_lenta_RJX1627	GL	2
SA252832	PTJ0295_3	PTJ0295	Eggerthella_lenta_RJX1627	GL	3
SA252833	PTJ0296_1	PTJ0296	Eggerthella_lenta_RJX1626	GL	1
SA252834	PTJ0296_2	PTJ0296	Eggerthella_lenta_RJX1626	GL	2
SA252835	PTJ0296_3	PTJ0296	Eggerthella_lenta_RJX1626	GL	3
SA252836	PTJ0297_1	PTJ0297	Eggerthella_lenta_SECOmt75m2	GL	1
SA252837	PTJ0297_2	PTJ0297	Eggerthella_lenta_SECOmt75m2	GL	2
SA252838	PTJ0297_3	PTJ0297	Eggerthella_lenta_SECOmt75m2	GL	3
SA252687	PTJ0181_3	-	-	-	-
SA252688	PTJ0181_2	-	-	-	-
SA252689	PTJ0174_2	-	-	-	-
SA252690	PTJ0182_1	-	-	-	-

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Collection:

Collection ID:	CO002603
Collection Summary:	For the comparative strain metabolomics experiment, 96-well polypropylene deep well plates were prepared with 800μL of fresh media in each well. Starter cultures and inocula for 29 isolates of Eggerthella lenta and 1 isolate of Eggerthella sinensis [26] were prepared as described above for growth assays, except without final dilution, and 80 μL was used to inoculate wells, leaving a blank well in between every culture well to prevent cross-contamination. After 72 hours, OD600 measurements were taken, plates were centrifuged, and supernatants were collected. 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:	single time point (72 hours)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002622
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:

TR002622

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:	SP002616
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:

SP002616

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	AN004133	AN004134
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:	CH003062
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:	MS003880
Analysis ID:	AN004133
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:	MS003881
Analysis ID:	AN004134
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