Summary of Study ST002515

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	ST002515
Study Title	Time course 2: Acetate quantification from growth of Eggerthella lenta in defined media
Study Type	Untargeted LC-MS
Study Summary	This dataset contains targeted metabolomics analysis of carboxylic acids in 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	2023-03-21
Raw Data Available	Yes
Raw Data File Type(s)	d, mzML
Analysis Type Detail	LC-MS
Release Date	2023-04-10
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:	SU002615
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	AcetateConc	TimePoint	Time
SA253097	P1-E02	2243	0	TP4	28.5
SA253098	P1-E05	2243	0	TP4	28.5
SA253099	P1-B03	2243	0	TP4	28.5
SA253100	P1-E03	2243	0	TP7	47.5
SA253101	P1-E07	2243	0	TP7	47.5
SA253102	P1-E09	2243	0	TP7	47.5
SA253103	P1-G05	2243	10	TP4	28.5
SA253104	P1-C02	2243	10	TP4	28.5
SA253105	P1-C05	2243	10	TP4	28.5
SA253106	P1-C09	2243	10	TP7	47.5
SA253107	P1-G07	2243	10	TP7	47.5
SA253108	P1-C07	2243	10	TP7	47.5
SA253109	P1-A11	2243	10	TP8	64
SA253110	P1-C11	2243	10	TP8	64
SA253111	P1-E11	2243	10	TP8	64
SA253112	P1-A02	2243	1	TP4	28.5
SA253113	P1-A05	2243	1	TP4	28.5
SA253114	P1-G02	2243	1	TP4	28.5
SA253115	P1-G09	2243	1	TP7	47.5
SA253116	P1-A09	2243	1	TP7	47.5
SA253117	P1-A07	2243	1	TP7	47.5
SA253118	P1-C10	2243	1	TP8	64
SA253119	P1-A10	2243	1	TP8	64
SA253120	P1-E10	2243	1	TP8	64
SA253121	P1-F02	AB8	0	TP4	28.5
SA253122	P1-F05	AB8	0	TP4	28.5
SA253123	P1-C03	AB8	0	TP4	28.5
SA253124	P1-F03	AB8	0	TP7	47.5
SA253125	P1-F09	AB8	0	TP7	47.5
SA253126	P1-F07	AB8	0	TP7	47.5
SA253127	P1-A12	AB8	10	TP4	28.5
SA253128	P1-D05	AB8	10	TP4	28.5
SA253129	P1-D02	AB8	10	TP4	28.5
SA253130	P1-D07	AB8	10	TP7	47.5
SA253131	P1-B12	AB8	10	TP7	47.5
SA253132	P1-D09	AB8	10	TP7	47.5
SA253133	P1-B11	AB8	10	TP8	64
SA253134	P1-D11	AB8	10	TP8	64
SA253135	P1-F11	AB8	10	TP8	64
SA253136	P1-B05	AB8	1	TP4	28.5
SA253137	P1-G03	AB8	1	TP4	28.5
SA253138	P1-B02	AB8	1	TP4	28.5
SA253139	P1-B07	AB8	1	TP7	47.5
SA253140	P1-C12	AB8	1	TP7	47.5
SA253141	P1-B09	AB8	1	TP7	47.5
SA253142	P1-B10	AB8	1	TP8	64
SA253143	P1-F10	AB8	1	TP8	64
SA253144	P1-D10	AB8	1	TP8	64
SA253065	Std_PBSSt_07	-	-	-	-
SA253066	Std_PBSSt_05	-	-	-	-
SA253067	Std_PBSSt_06	-	-	-	-
SA253068	Std_PBSSt_08	-	-	-	-
SA253069	Std_PBSSt_11	-	-	-	-
SA253070	Std_PBSSt_10	-	-	-	-
SA253071	Std_PBSSt_09	-	-	-	-
SA253072	Std_PBSSt_04	-	-	-	-
SA253073	Std_PBSSt_03	-	-	-	-
SA253074	PooledQC_01	-	-	-	-
SA253075	Blank_01	-	-	-	-
SA253076	PooledQC_02	-	-	-	-
SA253077	PooledQC_03	-	-	-	-
SA253078	Std_PBSSt_02	-	-	-	-
SA253079	Std_PBSSt_01	-	-	-	-
SA253080	Std_PBSSt_12	-	-	-	-
SA253081	Std_mSt_02	-	-	-	-
SA253082	Std_mSt_12	-	-	-	-
SA253083	Std_mSt_11	-	-	-	-
SA253084	Std_mSt_10	-	-	-	-
SA253085	Blank_05	-	-	-	-
SA253086	Blank_04	-	-	-	-
SA253087	Blank_02	-	-	-	-
SA253088	Blank_03	-	-	-	-
SA253089	Std_mSt_09	-	-	-	-
SA253090	Std_mSt_08	-	-	-	-
SA253091	Std_mSt_04	-	-	-	-
SA253092	Std_mSt_03	-	-	-	-
SA253093	Std_mSt_01	-	-	-	-
SA253094	Std_mSt_05	-	-	-	-
SA253095	Std_mSt_06	-	-	-	-
SA253096	Std_mSt_07	-	-	-	-
SA253163	P1-E04	c	0	TP4	-
SA253164	P1-E01	c	0	TP4	-
SA253165	P1-E08	c	0	TP7	-
SA253166	P1-E06	c	0	TP7	-
SA253167	P1-C04	c	10	TP4	-
SA253168	P1-C01	c	10	TP4	-
SA253169	P1-C06	c	10	TP7	-
SA253170	P1-C08	c	10	TP7	-
SA253171	P1-E12	c	10	TP8	-
SA253172	P1-G11	c	10	TP8	-
SA253173	P1-A01	c	1	TP4	-
SA253174	P1-A04	c	1	TP4	-
SA253175	P1-A06	c	1	TP7	-
SA253176	P1-A08	c	1	TP7	-
SA253177	P1-D12	c	1	TP8	-
SA253178	P1-G10	c	1	TP8	-
SA253145	P1-F04	Val	0	TP4	28.5
SA253146	P1-A03	Val	0	TP4	28.5
SA253147	P1-F01	Val	0	TP4	28.5
SA253148	P1-D03	Val	0	TP7	47.5

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Collection:

Collection ID:	CO002608
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 64 hours (full growth phase)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002627
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:

TR002627

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:	SP002621
Sampleprep Summary:	Samples (20 μL) were first mixed with an internal standard solution (30 μL; 1 mM phenylpropionate-d9) in a V-bottomed, poly(propylene), 96-well plate, and extracted by mixing with 3 sample volumes of extraction solution (75% acetonitrile:25% methanol). The plate was covered with a lid and centrifuged at 5,000 rcf for 15 min at 4 °C. Supernatant was collected for derivatization before subjecting to LC–MS analysis. Samples were processed using a derivatization method targeting compounds containing a free carboxylic acid. Extracted samples were mixed with 3-nitrophenylhydrazine (NPH; 200 mM in 50% acetonitrile) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (120 mM in 6% pyridine) at a 2:1:1 ratio. The plate was sealed with a plastic sealing mat (Thermo Fisher Scientific, #AB-0566) and incubated at 40 °C, 600 rpm in a thermomixer for 60 min to derivatize the carboxylate-containing compounds. The reaction mixture was quenched with 0.02% formic acid in 10% acetonitrile:water before LC–MS.

Sampleprep ID:

SP002621

Sampleprep Summary:

Samples (20 μL) were first mixed with an internal standard solution (30 μL; 1 mM phenylpropionate-d9) in a V-bottomed, poly(propylene), 96-well plate, and extracted by mixing with 3 sample volumes of extraction solution (75% acetonitrile:25% methanol). The plate was covered with a lid and centrifuged at 5,000 rcf for 15 min at 4 °C. Supernatant was collected for derivatization before subjecting to LC–MS analysis. Samples were processed using a derivatization method targeting compounds containing a free carboxylic acid. Extracted samples were mixed with 3-nitrophenylhydrazine (NPH; 200 mM in 50% acetonitrile) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (120 mM in 6% pyridine) at a 2:1:1 ratio. The plate was sealed with a plastic sealing mat (Thermo Fisher Scientific, #AB-0566) and incubated at 40 °C, 600 rpm in a thermomixer for 60 min to derivatize the carboxylate-containing compounds. The reaction mixture was quenched with 0.02% formic acid in 10% acetonitrile:water before LC–MS.

Combined analysis:

Analysis ID	AN004142
Analysis type	MS
Chromatography type	Unspecified
Chromatography system	Agilent 1290 Infinity II
Column	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Agilent 6545 QTOF
Ion Mode	NEGATIVE
Units	mM

Chromatography:

Chromatography ID:	CH003067
Chromatography Summary:	Samples were injected via refrigerated autosampler into mobile phase and chromatographically separated by an Agilent 1290 Infinity II UPLC and detected using an Agilent 6545XT Q-TOF (quadrupole time of flight) mass spectrometer equipped with a dual jet stream electrospray ionization source, operating under extended dynamic range (1,700 m/z). Chromatographic separation was performed using an ACQUITY Bridged Ethylene Hybrid (BEH) C18 column 2.1 x 100 mm, 1.7-micron particle size, (Waters Corp. Milford, MA), using chromatographic conditions published elsewhere (Supplementary Table S26 of https://doi.org/10.1038/s41564-022-01109-9).
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	50
Flow Gradient:	See
Flow Rate:	300 μL per minute
Solvent A:	0.1% formic acid in water
Solvent B:	0.1% formic acid in methanol
Chromatography Type:	Unspecified

MS:

MS ID:	MS003889
Analysis ID:	AN004142
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Samples were injected via refrigerated autosampler into mobile phase and chromatographically separated by an Agilent 1290 Infinity II UPLC and detected using an Agilent 6545XT Q-TOF (quadrupole time of flight) mass spectrometer equipped with a dual jet stream electrospray ionization source, operating under extended dynamic range (1,700 m/z). MS1 spectra were collected in centroid mode, and peak assignments in samples were made based on comparisons of retention times and accurate masses from authentic standards using MassHunter Quantitative Analysis v.10.0 software from Agilent Technologies. Acetate was quantified from calibration curves constructed with acetate-d4 as a standard using isotope-dilution MS with phenylpropionate-d9 as the internal standard. Calibration curves were performed in a modified base form of EDM1 lacking amino acids and other carboxylic acids. A background level of 1.05mM of acetate was subtracted to obtain the final quantities.
Ion Mode:	NEGATIVE