Summary of Study ST002519

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	ST002519
Study Title	Time course 3: Growth of Eggerthella lenta in defined media with some samples receiving 13C6 stable isotope labeled arginine (intracellular samples)
Study Type	Untargeted LC-MS
Study Summary	This dataset contains untargeted metabolomics analysis of supernatants from Eggerthella lenta grown in defined EDM1 media. One set of samples grew in EDM1 containing 13C6 stable isotope labeled arginine. 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	2023-04-09
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:	SU002619
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
SA253964	MSA0005	2243	HR	TP3	44
SA253965	MSA0003	2243	HR	TP3	44
SA253966	MSA0001	2243	HR	TP3	44
SA253967	MSA0018	2243	HR	TP4	56
SA253968	MSA0004	2243	HR	TP4	56
SA253969	MSA0009	2243	HR	TP4	56
SA253970	MSA0017	2243	R	TP3	44
SA253971	MSA0014	2243	R	TP3	44
SA253972	MSA0006	2243	R	TP3	44
SA253973	MSA0019	2243	R	TP4	56
SA253974	MSA0008	2243	R	TP4	56
SA253975	MSA0015	2243	R	TP4	56
SA253956	QC2	-	-	-	-
SA253957	QC4	-	-	-	-
SA253958	QC3	-	-	-	-
SA253959	BK1	-	-	-	-
SA253960	BK2	-	-	-	-
SA253961	BK3	-	-	-	-
SA253962	BK4	-	-	-	-
SA253963	QC1	-	-	-	-
SA253976	MSA0023	c	HR	TP3	44
SA253977	MSA0011	c	HR	TP3	44
SA253978	MSA0022	c	HR	TP3	44
SA253979	MSA0013	c	HR	TP4	56
SA253980	MSA0007	c	HR	TP4	56
SA253981	MSA0016	c	HR	TP4	56
SA253982	MSA0010	c	R	TP3	44
SA253983	MSA0012	c	R	TP3	44
SA253984	MSA0021	c	R	TP3	44
SA253985	MSA0002	c	R	TP4	56
SA253986	MSA0020	c	R	TP4	56
SA253987	MSA0024	c	R	TP4	56

Showing results 1 to 32 of 32

Showing results 1 to 32 of 32

Collection:

Collection ID:	CO002612
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 arginine in the defined media was replaced with 13C6 labeled arginine HCl (Sigma-Aldrich), 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:	TR002631
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:

TR002631

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:	SP002625
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:

SP002625

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	AN004149	AN004150
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:	CH003071
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:	MS003896
Analysis ID:	AN004149
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:	MS003897
Analysis ID:	AN004150
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