#METABOLOMICS WORKBENCH cnoecker_20230322_180807 DATATRACK_ID:3811 STUDY_ID:ST002519 ANALYSIS_ID:AN004150
VERSION                          	1
CREATED_ON                       	02-08-2024
#PROJECT
PR:PROJECT_TITLE                 	Systems biology illuminates the alternative metabolic niche of the human gut
PR:PROJECT_TITLE                 	bacterium Eggerthella lenta
PR:PROJECT_TYPE                  	Untargeted LC-MS
PR:PROJECT_SUMMARY               	Human gut bacteria perform diverse metabolic functions with consequences for
PR:PROJECT_SUMMARY               	host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta
PR:PROJECT_SUMMARY               	performs several unusual chemical transformations, but it does not metabolize
PR:PROJECT_SUMMARY               	sugars and its core growth strategy remains unclear. To obtain a comprehensive
PR:PROJECT_SUMMARY               	view of the metabolic network of E. lenta, we generated several complementary
PR:PROJECT_SUMMARY               	resources: defined culture media, metabolomics profiles of strain isolates, and
PR:PROJECT_SUMMARY               	a curated genome-scale metabolic reconstruction. Stable isotope-resolved
PR:PROJECT_SUMMARY               	metabolomics revealed that E. lenta uses acetate as a key carbon source while
PR:PROJECT_SUMMARY               	catabolizing arginine to generate ATP, traits which could be recapitulated in
PR:PROJECT_SUMMARY               	silico by our updated metabolic model. We compared these in vitro findings with
PR:PROJECT_SUMMARY               	metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying
PR:PROJECT_SUMMARY               	shared signatures across environments and highlighting catabolism of the host
PR:PROJECT_SUMMARY               	signaling metabolite agmatine as an alternative energy pathway. Together, our
PR:PROJECT_SUMMARY               	results elucidate a distinctive metabolic niche filled by E. lenta in the gut
PR:PROJECT_SUMMARY               	ecosystem.
PR:INSTITUTE                     	University of California, San Francisco
PR:DEPARTMENT                    	Microbiology and Immunology
PR:LABORATORY                    	Peter Turnbaugh
PR:LAST_NAME                     	Noecker
PR:FIRST_NAME                    	Cecilia
PR:ADDRESS                       	513 Parnassus Ave HSW1501, San Francisco, CA 94143
PR:EMAIL                         	cecilia.noecker@ucsf.edu
PR:PHONE                         	415-502-3264
PR:FUNDING_SOURCE                	This work was supported by the National Institutes of Health (2R01HL122593;
PR:FUNDING_SOURCE                	1R01AT011117; 1R01DK114034 to P.J.T., F32GM140808 to C.N.). P.J.T. is a Chan
PR:FUNDING_SOURCE                	Zuckerberg Biohub Investigator and held an Investigators in the Pathogenesis of
PR:FUNDING_SOURCE                	Infectious Disease Award from the Burroughs Wellcome Fund.
PR:PUBLICATIONS                  	https://doi.org/10.1101/2022.09.19.508335
PR:DOI                           	http://dx.doi.org/10.21228/M89B04
#STUDY
ST:STUDY_TITLE                   	Time course 3: Growth of Eggerthella lenta in defined media with some samples
ST:STUDY_TITLE                   	receiving 13C6 stable isotope labeled arginine (intracellular samples)
ST:STUDY_TYPE                    	Untargeted LC-MS
ST:STUDY_SUMMARY                 	This dataset contains untargeted metabolomics analysis of supernatants from
ST:STUDY_SUMMARY                 	Eggerthella lenta grown in defined EDM1 media. One set of samples grew in EDM1
ST:STUDY_SUMMARY                 	containing 13C6 stable isotope labeled arginine. Samples were collected at a
ST:STUDY_SUMMARY                 	subset of time points for extraction of intracellular metabolites.
ST:INSTITUTE                     	University of California, San Francisco
ST:LAST_NAME                     	Noecker
ST:FIRST_NAME                    	Cecilia
ST:ADDRESS                       	513 Parnassus Ave HSW1501, San Francisco, CA 94143
ST:EMAIL                         	cecilia.noecker@ucsf.edu
ST:PHONE                         	415-502-3264
ST:SUBMIT_DATE                   	2023-03-22
#SUBJECT
SU:SUBJECT_TYPE                  	Bacteria
SU:SUBJECT_SPECIES               	Eggerthella lenta
SU:TAXONOMY_ID                   	84112
SU:GENOTYPE_STRAIN               	DSM 2243
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	3	MSA0001	Strain:2243 | ArgGroup:HR | TimePoint:TP3 | Time:44	Sample=HR_2243_TP3i_3; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0001.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0001.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0001.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0001.mzML
SUBJECT_SAMPLE_FACTORS           	1	MSA0003	Strain:2243 | ArgGroup:HR | TimePoint:TP3 | Time:44	Sample=HR_2243_TP3i_1; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0003.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0003.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0003.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0003.mzML
SUBJECT_SAMPLE_FACTORS           	2	MSA0005	Strain:2243 | ArgGroup:HR | TimePoint:TP3 | Time:44	Sample=HR_2243_TP3i_2; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0005.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0005.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0005.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0005.mzML
SUBJECT_SAMPLE_FACTORS           	3	MSA0004	Strain:2243 | ArgGroup:HR | TimePoint:TP4 | Time:56	Sample=HR_2243_TP4i_3; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0004.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0004.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0004.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0004.mzML
SUBJECT_SAMPLE_FACTORS           	2	MSA0009	Strain:2243 | ArgGroup:HR | TimePoint:TP4 | Time:56	Sample=HR_2243_TP4i_2; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0009.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0009.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0009.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0009.mzML
SUBJECT_SAMPLE_FACTORS           	1	MSA0018	Strain:2243 | ArgGroup:HR | TimePoint:TP4 | Time:56	Sample=HR_2243_TP4i_1; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0018.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0018.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0018.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0018.mzML
SUBJECT_SAMPLE_FACTORS           	3	MSA0006	Strain:2243 | ArgGroup:R | TimePoint:TP3 | Time:44	Sample=R_2243_TP3i_3; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0006.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0006.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0006.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0006.mzML
SUBJECT_SAMPLE_FACTORS           	2	MSA0014	Strain:2243 | ArgGroup:R | TimePoint:TP3 | Time:44	Sample=R_2243_TP3i_2; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0014.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0014.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0014.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0014.mzML
SUBJECT_SAMPLE_FACTORS           	1	MSA0017	Strain:2243 | ArgGroup:R | TimePoint:TP3 | Time:44	Sample=R_2243_TP3i_1; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0017.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0017.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0017.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0017.mzML
SUBJECT_SAMPLE_FACTORS           	2	MSA0008	Strain:2243 | ArgGroup:R | TimePoint:TP4 | Time:56	Sample=R_2243_TP4i_2; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0008.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0008.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0008.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0008.mzML
SUBJECT_SAMPLE_FACTORS           	3	MSA0015	Strain:2243 | ArgGroup:R | TimePoint:TP4 | Time:56	Sample=R_2243_TP4i_3; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0015.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0015.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0015.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0015.mzML
SUBJECT_SAMPLE_FACTORS           	1	MSA0019	Strain:2243 | ArgGroup:R | TimePoint:TP4 | Time:56	Sample=R_2243_TP4i_1; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0019.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0019.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0019.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0019.mzML
SUBJECT_SAMPLE_FACTORS           	-	BK1	Strain:- | ArgGroup:- | TimePoint:- | Time:-	Sample=-; RAW_FILE_NAME=M00183_20220405Neg_BK1.raw; RAW_FILE_NAME=M00183_20220405Pos_BK1.raw; RAW_FILE_NAME=M00183_20220405Neg_BK1.mzML; RAW_FILE_NAME=M00183_20220405Pos_BK1.mzML
SUBJECT_SAMPLE_FACTORS           	-	BK2	Strain:- | ArgGroup:- | TimePoint:- | Time:-	Sample=-; RAW_FILE_NAME=M00183_20220405Neg_BK2.raw; RAW_FILE_NAME=M00183_20220405Pos_BK2.raw; RAW_FILE_NAME=M00183_20220405Neg_BK2.mzML; RAW_FILE_NAME=M00183_20220405Pos_BK2.mzML
SUBJECT_SAMPLE_FACTORS           	-	BK3	Strain:- | ArgGroup:- | TimePoint:- | Time:-	Sample=-; RAW_FILE_NAME=M00183_20220405Neg_BK3.raw; RAW_FILE_NAME=M00183_20220405Pos_BK3.raw; RAW_FILE_NAME=M00183_20220405Neg_BK3.mzML; RAW_FILE_NAME=M00183_20220405Pos_BK3.mzML
SUBJECT_SAMPLE_FACTORS           	-	BK4	Strain:- | ArgGroup:- | TimePoint:- | Time:-	Sample=-; RAW_FILE_NAME=M00183_20220405Neg_BK4.raw; RAW_FILE_NAME=M00183_20220405Pos_BK4.raw; RAW_FILE_NAME=M00183_20220405Neg_BK4.mzML; RAW_FILE_NAME=M00183_20220405Pos_BK4.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC1	Strain:- | ArgGroup:- | TimePoint:- | Time:-	Sample=-; RAW_FILE_NAME=M00183_20220405Neg_QC1.raw; RAW_FILE_NAME=M00183_20220405Pos_QC1.raw; RAW_FILE_NAME=M00183_20220405Neg_QC1.mzML; RAW_FILE_NAME=M00183_20220405Pos_QC1.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC2	Strain:- | ArgGroup:- | TimePoint:- | Time:-	Sample=-; RAW_FILE_NAME=M00183_20220405Neg_QC2.raw; RAW_FILE_NAME=M00183_20220405Pos_QC2.raw; RAW_FILE_NAME=M00183_20220405Neg_QC2.mzML; RAW_FILE_NAME=M00183_20220405Pos_QC2.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC3	Strain:- | ArgGroup:- | TimePoint:- | Time:-	Sample=-; RAW_FILE_NAME=M00183_20220405Neg_QC3.raw; RAW_FILE_NAME=M00183_20220405Pos_QC3.raw; RAW_FILE_NAME=M00183_20220405Neg_QC3.mzML; RAW_FILE_NAME=M00183_20220405Pos_QC3.mzML
SUBJECT_SAMPLE_FACTORS           	-	QC4	Strain:- | ArgGroup:- | TimePoint:- | Time:-	Sample=-; RAW_FILE_NAME=M00183_20220405Neg_QC4.raw; RAW_FILE_NAME=M00183_20220405Pos_QC4.raw; RAW_FILE_NAME=M00183_20220405Neg_QC4.mzML; RAW_FILE_NAME=M00183_20220405Pos_QC4.mzML
SUBJECT_SAMPLE_FACTORS           	1	MSA0011	Strain:c | ArgGroup:HR | TimePoint:TP3 | Time:44	Sample=HR_c_TP3i_1; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0011.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0011.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0011.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0011.mzML
SUBJECT_SAMPLE_FACTORS           	2	MSA0022	Strain:c | ArgGroup:HR | TimePoint:TP3 | Time:44	Sample=HR_c_TP3i_2; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0022.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0022.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0022.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0022.mzML
SUBJECT_SAMPLE_FACTORS           	3	MSA0023	Strain:c | ArgGroup:HR | TimePoint:TP3 | Time:44	Sample=HR_c_TP3i_3; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0023.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0023.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0023.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0023.mzML
SUBJECT_SAMPLE_FACTORS           	3	MSA0007	Strain:c | ArgGroup:HR | TimePoint:TP4 | Time:56	Sample=HR_c_TP4i_3; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0007.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0007.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0007.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0007.mzML
SUBJECT_SAMPLE_FACTORS           	2	MSA0013	Strain:c | ArgGroup:HR | TimePoint:TP4 | Time:56	Sample=HR_c_TP4i_2; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0013.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0013.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0013.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0013.mzML
SUBJECT_SAMPLE_FACTORS           	1	MSA0016	Strain:c | ArgGroup:HR | TimePoint:TP4 | Time:56	Sample=HR_c_TP4i_1; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0016.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0016.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0016.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0016.mzML
SUBJECT_SAMPLE_FACTORS           	2	MSA0010	Strain:c | ArgGroup:R | TimePoint:TP3 | Time:44	Sample=R_c_TP3i_2; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0010.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0010.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0010.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0010.mzML
SUBJECT_SAMPLE_FACTORS           	3	MSA0012	Strain:c | ArgGroup:R | TimePoint:TP3 | Time:44	Sample=R_c_TP3i_3; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0012.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0012.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0012.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0012.mzML
SUBJECT_SAMPLE_FACTORS           	1	MSA0021	Strain:c | ArgGroup:R | TimePoint:TP3 | Time:44	Sample=R_c_TP3i_1; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0021.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0021.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0021.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0021.mzML
SUBJECT_SAMPLE_FACTORS           	2	MSA0002	Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56	Sample=R_c_TP4i_2; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0002.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0002.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0002.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0002.mzML
SUBJECT_SAMPLE_FACTORS           	3	MSA0020	Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56	Sample=R_c_TP4i_3; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0020.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0020.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0020.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0020.mzML
SUBJECT_SAMPLE_FACTORS           	1	MSA0024	Strain:c | ArgGroup:R | TimePoint:TP4 | Time:56	Sample=R_c_TP4i_1; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0024.raw; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0024.raw; RAW_FILE_NAME=M00183_20220405_Neg_1_MSA0024.mzML; RAW_FILE_NAME=M00183_20220405_Pos_1_MSA0024.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Time course experiments were conducted in tubes in the anaerobic chamber in a
CO:COLLECTION_SUMMARY            	37°C incubator. For all metabolomics experiments, three independent culture
CO:COLLECTION_SUMMARY            	replicates were included for each condition, with an equal number of
CO:COLLECTION_SUMMARY            	uninoculated control tubes. Starter cultures and inocula were prepared as
CO:COLLECTION_SUMMARY            	described above for growth assays. 5mLs of defined media was added to VWR glass
CO:COLLECTION_SUMMARY            	culture tubes (53283-800) with screw caps. The PBS-washed inoculum was added to
CO:COLLECTION_SUMMARY            	culture tubes to obtain an approximate starting OD600 of 0.001. A preliminary
CO:COLLECTION_SUMMARY            	growth assay was conducted to define time points spanning the exponential growth
CO:COLLECTION_SUMMARY            	phase in the tested conditions. At each time point, OD600 measurements of all
CO:COLLECTION_SUMMARY            	inoculated tubes were first measured using a Hach DR1900 spectrophotometer, with
CO:COLLECTION_SUMMARY            	a paired control tube to normalize for the background. 100 μL from each tube
CO:COLLECTION_SUMMARY            	were then transferred into a 96-well microplate, which was sealed and removed
CO:COLLECTION_SUMMARY            	from the anaerobic chamber. Plates were centrifuged at 1,928 rcf at 4°C for 8
CO:COLLECTION_SUMMARY            	minutes, after which supernatants were collected into fresh polypropylene tubes
CO:COLLECTION_SUMMARY            	or plates, sealed, and flash-frozen in liquid nitrogen. Two time course
CO:COLLECTION_SUMMARY            	experiments were carried out with stable isotope-labeled substrates.
CO:COLLECTION_SUMMARY            	Experimental groups included conditions in which arginine in the defined media
CO:COLLECTION_SUMMARY            	was replaced with 13C6 labeled arginine HCl (Sigma-Aldrich), along with a
CO:COLLECTION_SUMMARY            	matched experimental group with the same concentration of unlabeled substrate.
CO:COLLECTION_SUMMARY            	Intracellular extract samples were prepared with the following procedure, which
CO:COLLECTION_SUMMARY            	was optimized for lysis of thick gram-positive cell walls: 600 μL of culture
CO:COLLECTION_SUMMARY            	was transferred to an Eppendorf tube in anaerobic conditions and subsequently
CO:COLLECTION_SUMMARY            	centrifuged at 10,000rcf for three minutes at 4°C, after which the supernatant
CO:COLLECTION_SUMMARY            	was removed and the samples were immediately flash frozen to quench metabolites.
CO:COLLECTION_SUMMARY            	300 μL of cold methanol was then added to each pellet, followed by sonication
CO:COLLECTION_SUMMARY            	on ice for 5 minutes and then shaking at 4°C for 4-12 hours. Samples were then
CO:COLLECTION_SUMMARY            	centrifuged at 4°C at 15,000 rcf for 8 minutes, after which 120 μL of
CO:COLLECTION_SUMMARY            	supernatant was transferred to fresh tubes and stored at -80°C until analysis.
CO:SAMPLE_TYPE                   	Bacterial culture supernatant
CO:COLLECTION_FREQUENCY          	at time points specified in study design table over 64 hours (full growth phase)
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	For growth and metabolomics experiments, glycerol stocks of the 3 E. lenta
TR:TREATMENT_SUMMARY             	strains were first streaked on BHI+ agar plates and incubated at 37°C for 2-3
TR:TREATMENT_SUMMARY             	days. Individual colonies were inoculated into 3-4 mL liquid BHI+ and incubated
TR:TREATMENT_SUMMARY             	at 37°C for 40-48 hours, or until approximately early stationary phase. Culture
TR:TREATMENT_SUMMARY             	optical density (600 nm wavelength absorbance, OD600) was measured using a Hach
TR:TREATMENT_SUMMARY             	DR1900 spectrophotometer. 1 mL samples of BHI starter cultures were then
TR:TREATMENT_SUMMARY             	centrifuged at 1,568 rcf for 4 minutes in a microcentrifuge (ThermoScientific
TR:TREATMENT_SUMMARY             	mySpin 12) in the anaerobic chamber and resuspended in 1 mL sterile
TR:TREATMENT_SUMMARY             	phosphate-buffered saline (PBS). The resulting suspension was vortexed and
TR:TREATMENT_SUMMARY             	diluted to an approximate OD600 of 0.1, and used as inoculum into defined
TR:TREATMENT_SUMMARY             	experimental conditions. Varying media conditions were prepared separately and
TR:TREATMENT_SUMMARY             	all allowed to fully reduce in the anaerobic chamber prior to inoculation.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Prior to analysis, intracellular samples were dried at room temperature via
SP:SAMPLEPREP_SUMMARY            	Centrivap Benchtop Concentrator (Labconco Corp.). Samples were re-suspended in
SP:SAMPLEPREP_SUMMARY            	60 μL of a chilled solution of 1:1 methanol and acetonitrile, with 24% water at
SP:SAMPLEPREP_SUMMARY            	-20oC containing the internal standards CUDA and VAL-TYR-VAL each at 60 ng/mL.
SP:SAMPLEPREP_SUMMARY            	Samples were centrifuged at 4°C, 4,122 rcf for 5 minutes and the supernatant
SP:SAMPLEPREP_SUMMARY            	transferred to a vial and immediately capped for LC-MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Samples, sterile media, pools, and blanks were promptly added to a Thermo
CH:CHROMATOGRAPHY_SUMMARY        	Vanquish Autosampler at 4°C in a Vanquish UHPLC (Thermo Fisher Scientific,
CH:CHROMATOGRAPHY_SUMMARY        	Waltham, MA). Chromatographic separation was performed using an ACQUITY Bridged
CH:CHROMATOGRAPHY_SUMMARY        	Ethylene Hybrid (BEH) Amide column 2.1 x 150 mm, 1.7-micron particle size,
CH:CHROMATOGRAPHY_SUMMARY        	(Waters Corp. Milford, MA), using chromatographic conditions published elsewhere
CH:CHROMATOGRAPHY_SUMMARY        	(HILIC method described in the Supplementary Methods of
CH:CHROMATOGRAPHY_SUMMARY        	doi.org/10.1038/s41586-021-03707-9).
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
CH:COLUMN_TEMPERATURE            	40
CH:FLOW_GRADIENT                 	The gradient profile was held at 100% B for 2 minutes, from 100% B to 70% B in 5
CH:FLOW_GRADIENT                 	minutes, holding at 70% B for 0.7 minute, from 70% B to 40% B for 1.3 minutes,
CH:FLOW_GRADIENT                 	holding at 40% B for 0.5 minutes, from 40% B to 30% B for 0.75 minutes, before
CH:FLOW_GRADIENT                 	returning to 100% B for 2.5 minutes and holding at 100% B for 4 minutes.
CH:FLOW_RATE                     	400 μL per minute
CH:SOLVENT_A                     	100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3
CH:SOLVENT_B                     	95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate
CH:CHROMATOGRAPHY_TYPE           	HILIC
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF hybrid Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2
MS:MS_COMMENTS                   	selection of metabolites from our in-house ‘local’ library, when additional
MS:MS_COMMENTS                   	scan bandwidth was available MS2 was collected in a data-dependent manner. Mass
MS:MS_COMMENTS                   	range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was
MS:MS_COMMENTS                   	collected, loop count was 4, isolation window was 1.5 Da. In SIRM samples,
MS:MS_COMMENTS                   	deuterated internal standards were replaced with CUDA and Val-Tyr-Val to enable
MS:MS_COMMENTS                   	untargeted enrichment analysis. LC-MS/MS analysis conditions for SIRM
MS:MS_COMMENTS                   	metabolomics were identical to those used for standard untargeted metabolomics.
MS:MS_COMMENTS                   	Intra- and extracellular untargeted data generated from SIRM experiments was
MS:MS_COMMENTS                   	analyzed separately using Compound Discoverer version 3.3 (Thermo Scientific,
MS:MS_COMMENTS                   	Bremen, Germany). Samples treated with labeled compounds were always paired with
MS:MS_COMMENTS                   	matched samples treated with unlabeled compounds in order to correct for
MS:MS_COMMENTS                   	naturally occurring isotope abundances. Unlabeled samples were used for compound
MS:MS_COMMENTS                   	detection and formula assignment via isotope pattern-based prediction, spectral
MS:MS_COMMENTS                   	library matches, or mass lists matches. The isotope patterns and formulas from
MS:MS_COMMENTS                   	the sample files then served as a reference for the detection of potential
MS:MS_COMMENTS                   	isotopologues per compound in the labeled sample type. A specification of the
MS:MS_COMMENTS                   	full Compound Discoverer workflow is available at
MS:MS_COMMENTS                   	https://github.com/turnbaughlab/2022_Noecker_ElentaMetabolism.
MS:ION_MODE                      	NEGATIVE
#END