Summary of Study ST002518

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	ST002518
Study Title	Time course 3: Growth of Eggerthella lenta in defined media with some samples receiving 13C6 stable isotope labeled arginine
Study Type	Untargeted LC-MS
Study Summary	This dataset contains untargeted metabolomics analysis of supernatants from Eggerthella lenta DSM 2243 grown in defined EDM1 media. One set of samples grew in EDM1 containing 13C6 stable isotope labeled arginine.
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	2023-04-09
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:	SU002618
Subject Type:	Bacteria
Subject Species:	Eggerthella lenta
Taxonomy ID:	84112
Genotype Strain:	DSM 2243

Factors:

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

mb_sample_id	local_sample_id	Strain	ArgGroup	TimePoint	Time
SA253892	MSA0050	2243	HR	TP0	0
SA253893	MSA0043	2243	HR	TP0	0
SA253894	MSA0029	2243	HR	TP0	0
SA253895	MSA0049	2243	HR	TP1	20
SA253896	MSA0056	2243	HR	TP1	20
SA253897	MSA0004	2243	HR	TP1	20
SA253898	MSA0032	2243	HR	TP2	32
SA253899	MSA0031	2243	HR	TP2	32
SA253900	MSA0064	2243	HR	TP2	32
SA253901	MSA0007	2243	HR	TP3	44
SA253902	MSA0014	2243	HR	TP3	44
SA253903	MSA0025	2243	HR	TP3	44
SA253904	MSA0053	2243	HR	TP4	56
SA253905	MSA0044	2243	HR	TP4	56
SA253906	MSA0060	2243	HR	TP4	56
SA253907	MSA0055	2243	R	TP0	0
SA253908	MSA0054	2243	R	TP0	0
SA253909	MSA0013	2243	R	TP0	0
SA253910	MSA0048	2243	R	TP1	20
SA253911	MSA0033	2243	R	TP1	20
SA253912	MSA0018	2243	R	TP1	20
SA253913	MSA0042	2243	R	TP2	32
SA253914	MSA0017	2243	R	TP2	32
SA253915	MSA0026	2243	R	TP2	32
SA253916	MSA0039	2243	R	TP3	44
SA253917	MSA0008	2243	R	TP3	44
SA253918	MSA0009	2243	R	TP3	44
SA253919	MSA0034	2243	R	TP4	56
SA253920	MSA0001	2243	R	TP4	56
SA253921	MSA0002	2243	R	TP4	56
SA253878	QC5	-	-	-	-
SA253879	QC6	-	-	-	-
SA253880	QC4	-	-	-	-
SA253881	QC3	-	-	-	-
SA253882	BK1	-	-	-	-
SA253883	QC2	-	-	-	-
SA253884	QC7	-	-	-	-
SA253885	QC1	-	-	-	-
SA253886	BK4	-	-	-	-
SA253887	BK3	-	-	-	-
SA253888	BK7	-	-	-	-
SA253889	BK5	-	-	-	-
SA253890	BK2	-	-	-	-
SA253891	BK6	-	-	-	-
SA253922	MSA0059	c	HR	TP0	0
SA253923	MSA0028	c	HR	TP0	0
SA253924	MSA0062	c	HR	TP0	0
SA253925	MSA0046	c	HR	TP1	20
SA253926	MSA0012	c	HR	TP1	20
SA253927	MSA0024	c	HR	TP1	20
SA253928	MSA0057	c	HR	TP2	32
SA253929	MSA0038	c	HR	TP2	32
SA253930	MSA0030	c	HR	TP2	32
SA253931	MSA0035	c	HR	TP3	44
SA253932	MSA0016	c	HR	TP3	44
SA253933	MSA0027	c	HR	TP3	44
SA253934	MSA0015	c	HR	TP4	56
SA253935	MSA0047	c	HR	TP4	56
SA253936	MSA0036	c	HR	TP4	56
SA253937	MSA0061	c	R	TP0	0
SA253938	MSA0045	c	R	TP0	0
SA253939	MSA0052	c	R	TP0	0
SA253940	MSA0041	c	R	TP1	20
SA253941	MSA0021	c	R	TP1	20
SA253942	MSA0010	c	R	TP1	20
SA253943	MSA0019	c	R	TP2	32
SA253944	MSA0023	c	R	TP2	32
SA253945	MSA0022	c	R	TP2	32
SA253946	MSA0006	c	R	TP3	44
SA253947	MSA0058	c	R	TP3	44
SA253948	MSA0020	c	R	TP3	44
SA253949	MSA0037	c	R	TP4	56
SA253950	MSA0040	c	R	TP4	56
SA253951	MSA0003	c	R	TP4	56
SA253952	MSA0051	c	R	TP4	56
SA253953	MSA0011	c	R	TP4	56
SA253954	MSA0063	c	R	TP4	56
SA253955	MSA0005	c	R	TP4	56

Showing results 1 to 78 of 78

Showing results 1 to 78 of 78

Collection:

Collection ID:	CO002611
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 arginine in the defined media was replaced with 13C6 labeled arginine HCl, 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 56 hours (full growth phase)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002630
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:

TR002630

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:	SP002624
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:

SP002624

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	AN004147	AN004148
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:	CH003070
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:	MS003894
Analysis ID:	AN004147
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:	MS003895
Analysis ID:	AN004148
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