Summary of Study ST004535

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 PR002859. The data can be accessed directly via it's Project DOI: 10.21228/M83V8G This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004535
Study Title	Extracellular and Intracellular DCA derivatives level alteration
Study Summary	We investigated metabolic interactions between Bacteroides thetaiotaomicron (DSMZ 2079; BT) and Collinsella aerofaciens (DSMZ 3979; CA) by coculturing the two species under deoxycholic acid (DCA) stress alongside untreated controls. Cultures were grown in five replicates anaerobically in Brain Heart Infusion–supplemented (BHIS) medium at 37°C for 6 h with shaking, using Hungate tubes supplemented with 300 µM DCA where indicated. Across these conditions, we quantified extracellular amino acids, short-chain fatty acids, vitamin B6, and both extracellular and intracellular DCA derivatives to capture key metabolic responses to bile acid stress and microbial interactions. This section focuses on changes in extracellular and intracellular DCA derivatives profiles, providing an overview of how bile acid stress this two-member community. In anaerobic coculture, C. aerofaciens mitigated the inhibitory effects of deoxycholic acid (DCA) on B. thetaiotaomicron by absorbing DCA from the medium and partially converting it into glycine-conjugated derivatives. These results suggest that microbial interactions can enhance resistance to bile acid stress and may influence gut microbiome resilience, with potential relevance for liver- and bile acid–related disorders. CA: Collinsella aerofaciens; BT: Bacteroides thetaiotaomicron; Co: Coculture of the two species
Institute	Charles University
Department	Institute of Medical Biochemistry and Laboratory Diagnostics
Last Name	Leníček
First Name	Martin
Address	Katerinska 32, 12108 Prague, Czech Republic
Email	Martin.Lenicek@lf1.cuni.cz
Phone	+420723748496
Submit Date	2026-01-08
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2026-01-19
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002859
Project DOI:	doi: 10.21228/M83V8G
Project Title:	Coculture of Collinsella aerofaciens and Bacteroides thetaiotaomicron under bile acid stress reveals vitamin B6 exchange
Project Summary:	Background: Although the bile acid-mediated microbiome-host interaction is known to shape both the composition and functionality of the gut microbiome, the mechanisms by which bile acid stress influences specific microbial metabolic interactions remain poorly understood. To address this gap, we examine the metabolic interplay between two key gut microbes. Bacteroides thetaiotaomicron, one of the most abundant species, possesses a broad enzymatic repertoire for polysaccharide degradation, while Collinsella aerofaciens is associated with liver-related diseases and plays a role in modifying primary bile acids. Results: In anaerobic coculture, C. aerofaciens mitigated the inhibitory effects of deoxycholic acid (DCA) on B. thetaiotaomicron by absorbing DCA from the medium and partially converting it into glycine-conjugated derivatives. Proteomic analysis showed that DCA broadly disrupted amino acid and vitamin metabolism pathways, particularly in B. thetaiotaomicron. In contrast, coculture led to a general upregulation of these pathways in C. aerofaciens, with a marked activation of vitamin B6 metabolism. Additionally, C. aerofaciens exhibited increased production of citrulline and ornithine in coculture. Conclusions: C. aerofaciens alleviates DCA toxicity on B. thetaiotaomicron through absorption, while promoting amino acid and vitamin metabolism, including the vitamin B6 synthesis pathway, during coculture. These results suggest that microbial interactions can enhance resistance to bile acid stress and may influence gut microbiome resilience, with potential relevance for liver- and bile acid–related disorders.
Institute:	Helmholtz Centre for Environmental Research
Department:	Department of Molecular Toxicology
Laboratory:	Functional Metabolomics
Last Name:	Wang
First Name:	Yan
Address:	Permoserstraße 15, 04318, Leipzig, Germany
Email:	yan.wang@ufz.de
Phone:	+49 341 60251532

Subject:

Subject ID:	SU004714
Subject Type:	Bacteria
Subject Species:	Bacteroides thetaiotaomicron,Collinsella aerofaciens
Taxonomy ID:	818,74426
Genotype Strain:	Bacteroides thetaiotaomicron (DSMZ 2079; BT) and Collinsella aerofaciens (DSMZ 3979; CA)

Factors:

Subject type: Bacteria; Subject species: Bacteroides thetaiotaomicron,Collinsella aerofaciens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment	Replicate	Sample Type
SA537864	BT_B1_P	BT	Control	1	Pellet
SA537865	BT_B1_SN	BT	Control	1	Supernatant
SA537866	BT_B2_P	BT	Control	2	Pellet
SA537867	BT_B2_SN	BT	Control	2	Supernatant
SA537868	BT_B3_P	BT	Control	3	Pellet
SA537869	BT_B3_SN	BT	Control	3	Supernatant
SA537870	BT_B4_P	BT	Control	4	Pellet
SA537871	BT_B4_SN	BT	Control	4	Supernatant
SA537872	BT_B5_P	BT	Control	5	Pellet
SA537873	BT_B5_SN	BT	Control	5	Supernatant
SA537874	BT_DCA1_P	BT	final concentration 300µM DCA	1	Pellet
SA537875	BT_DCA1_SN	BT	final concentration 300µM DCA	1	Supernatant
SA537876	BT_DCA2_P	BT	final concentration 300µM DCA	2	Pellet
SA537877	BT_DCA2_SN	BT	final concentration 300µM DCA	2	Supernatant
SA537878	BT_DCA3_P	BT	final concentration 300µM DCA	3	Pellet
SA537879	BT_DCA3_SN	BT	final concentration 300µM DCA	3	Supernatant
SA537880	BT_DCA4_P	BT	final concentration 300µM DCA	4	Pellet
SA537881	BT_DCA4_SN	BT	final concentration 300µM DCA	4	Supernatant
SA537882	BT_DCA5_P	BT	final concentration 300µM DCA	5	Pellet
SA537883	BT_DCA5_SN	BT	final concentration 300µM DCA	5	Supernatant
SA537884	CA_B1_P	CA	Control	1	Pellet
SA537885	CA_B1_SN	CA	Control	1	Supernatant
SA537886	CA_B2_P	CA	Control	2	Pellet
SA537887	CA_B2_SN	CA	Control	2	Supernatant
SA537888	CA_B3_P	CA	Control	3	Pellet
SA537889	CA_B3_SN	CA	Control	3	Supernatant
SA537890	CA_B4_P	CA	Control	4	Pellet
SA537891	CA_B4_SN	CA	Control	4	Supernatant
SA537892	CA_B5_P	CA	Control	5	Pellet
SA537893	CA_B5_SN	CA	Control	5	Supernatant
SA537894	CA_DCA1_P	CA	final concentration 300µM DCA	1	Pellet
SA537895	CA_DCA1_SN	CA	final concentration 300µM DCA	1	Supernatant
SA537896	CA_DCA2_P	CA	final concentration 300µM DCA	2	Pellet
SA537897	CA_DCA2_SN	CA	final concentration 300µM DCA	2	Supernatant
SA537898	CA_DCA3_P	CA	final concentration 300µM DCA	3	Pellet
SA537899	CA_DCA3_SN	CA	final concentration 300µM DCA	3	Supernatant
SA537900	CA_DCA4_P	CA	final concentration 300µM DCA	4	Pellet
SA537901	CA_DCA4_SN	CA	final concentration 300µM DCA	4	Supernatant
SA537902	CA_DCA5_P	CA	final concentration 300µM DCA	5	Pellet
SA537903	CA_DCA5_SN	CA	final concentration 300µM DCA	5	Supernatant
SA537904	Co_B1_P	Co	Control	1	Pellet
SA537905	Co_B1_SN	Co	Control	1	Supernatant
SA537906	Co_B2_P	Co	Control	2	Pellet
SA537907	Co_B2_SN	Co	Control	2	Supernatant
SA537908	Co_B3_P	Co	Control	3	Pellet
SA537909	Co_B3_SN	Co	Control	3	Supernatant
SA537910	Co_B4_P	Co	Control	4	Pellet
SA537911	Co_B4_SN	Co	Control	4	Supernatant
SA537912	Co_B5_SN	Co	Control	5	Supernatant
SA537913	Co_DCA1_SN	Co	final concentration 300µM DCA	1	Supernatant
SA537914	Co_DCA2_SN	Co	final concentration 300µM DCA	2	Supernatant
SA537915	Co_DCA3_SN	Co	final concentration 300µM DCA	3	Supernatant
SA537916	Co_DCA4_P	Co	final concentration 300µM DCA	4	Pellet
SA537917	Co_DCA4_SN	Co	final concentration 300µM DCA	4	Supernatant
SA537918	Co_DCA5_P	Co	final concentration 300µM DCA	5	Pellet
SA537919	Co_DCA5_SN	Co	final concentration 300µM DCA	5	Supernatant
SA537920	Blank_SN	NA	Blank	1	Supernatant
SA537921	300 µM DCA_SN	NA	Blank	1	Supernatant

Showing results 1 to 58 of 58

Showing results 1 to 58 of 58

Collection:

Collection ID:	CO004707
Collection Summary:	After 6 hours of incubation, 2.5 mL of bacterial suspension was harvested. 2 mL of that was centrifuged at 4°C for 10 minutes to separate the supernatant and pellet. Both fractions were immediately frozen at –80°C for further omics analysis.
Sample Type:	Bacterial culture supernatant & pellet

Treatment:

Treatment ID:	TR004723
Treatment Summary:	Hungate anaerobic culture tubes containing BHIS medium were supplemented with DCA to a final concentration of 300 µM and flushed with pure nitrogen gas. Strains were then inoculated into the tubes in five replicates each. Cultures were monitored at 600 nm using a Nanocolour® UV/VIS II spectrophotometer (Macherey-Nagel). After 6 hours of incubation, 2.5 mL of bacterial suspension was harvested. 2 mL of that was centrifuged at 4°C for 10 minutes to separate the supernatant and pellet. Both fractions were immediately frozen at –80°C for further omics analysis.

Treatment ID:

TR004723

Treatment Summary:

Hungate anaerobic culture tubes containing BHIS medium were supplemented with DCA to a final concentration of 300 µM and flushed with pure nitrogen gas. Strains were then inoculated into the tubes in five replicates each. Cultures were monitored at 600 nm using a Nanocolour® UV/VIS II spectrophotometer (Macherey-Nagel). After 6 hours of incubation, 2.5 mL of bacterial suspension was harvested. 2 mL of that was centrifuged at 4°C for 10 minutes to separate the supernatant and pellet. Both fractions were immediately frozen at –80°C for further omics analysis.

Sample Preparation:

Sampleprep ID:	SP004720
Sampleprep Summary:	Intracellular DCA and derivatives were extracted from cell pellets kept on ice. Pellets were resuspended in 500 µL of pre-cooled (–20°C) methanol and subjected to three freeze–thaw cycles in liquid nitrogen. To ensure cell disruption, suspensions were sonicated for 10 min. After centrifugation at 16,000 g for 5 min at –4°C, the supernatant was transferred to a fresh tube. The pellet was re-extracted using the same procedure, and both supernatants were combined and dried completely in an Eppendorf® Concentrator Plus at room temperature. Dried extracts were stored at –80°C until analysis. The supernatant was collected and stored at –80°C until bile acid analysis. The dried residue was reconstituted in 300 µL of methanol. For DCA quantification, 3 µL of this solution was analysed, while 250 µL were used for the quantification of glyco-, tauro-, and keto-derivatives of DCA. Each aliquot was spiked with an internal standard mixture (d4-DCA, d4-UDCA, d4-gCDCA), evaporated to dryness, and reconstituted in 100 µL of mobile phase.

Sampleprep ID:

SP004720

Sampleprep Summary:

Intracellular DCA and derivatives were extracted from cell pellets kept on ice. Pellets were resuspended in 500 µL of pre-cooled (–20°C) methanol and subjected to three freeze–thaw cycles in liquid nitrogen. To ensure cell disruption, suspensions were sonicated for 10 min. After centrifugation at 16,000 g for 5 min at –4°C, the supernatant was transferred to a fresh tube. The pellet was re-extracted using the same procedure, and both supernatants were combined and dried completely in an Eppendorf® Concentrator Plus at room temperature. Dried extracts were stored at –80°C until analysis. The supernatant was collected and stored at –80°C until bile acid analysis. The dried residue was reconstituted in 300 µL of methanol. For DCA quantification, 3 µL of this solution was analysed, while 250 µL were used for the quantification of glyco-, tauro-, and keto-derivatives of DCA. Each aliquot was spiked with an internal standard mixture (d4-DCA, d4-UDCA, d4-gCDCA), evaporated to dryness, and reconstituted in 100 µL of mobile phase.

Combined analysis:

Analysis ID	AN007617	AN007618
Chromatography ID	CH005777	CH005778
MS ID	MS007314	MS007315
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Dionex Ultimate 3000	Dionex Ultimate 3000
Column	Pinnacle DB C18 column (100 × 2.1 mm, 3 μm)	Pinnacle DB C18 column (100 × 2.1 mm, 3 μm)
MS Type	ESI	ESI
MS instrument type	triple quadrupole	triple quadrupole
MS instrument name	TSQ Quantum Access Max with H-ESI II probe	TSQ Quantum Access Max with H-ESI II probe
Ion Mode	NEGATIVE	NEGATIVE
Units	µM	µM

Chromatography:

Chromatography ID:	CH005777
Instrument Name:	Dionex Ultimate 3000
Column Name:	Pinnacle DB C18 column (100 × 2.1 mm, 3 μm)
Column Temperature:	55°C
Flow Gradient:	0–2.5 min 40%; 2.5–3.5 min 40–57%; 3.5–9.5 min 57–59%; 9.5–10.0 min 59–70%; 10.0–14.0 min 70–72%; 14.0–16.0 min 72–76%.
Flow Rate:	0.3 mL/min
Solvent A:	100% Water; 0.005 M Ammonium acetate; 0.012% formic acid
Solvent B:	100% Methanol
Chromatography Type:	Reversed phase

Chromatography ID:	CH005778
Instrument Name:	Dionex Ultimate 3000
Column Name:	Pinnacle DB C18 column (100 × 2.1 mm, 3 μm)
Column Temperature:	55°C
Flow Gradient:	0–2.5 min 40%; 2.5–3.5 min 40–57%; 3.5–9.5 min 57–59%; 9.5–10.0 min 59–70%; 10.0–14.0 min 70–72%; 14.0–16.0 min 72–76%.
Flow Rate:	0.3 mL/min
Solvent A:	100% Water; 0.005 M Ammonium acetate; 0.012% formic acid
Solvent B:	100% Methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS007314
Analysis ID:	AN007617
Instrument Name:	TSQ Quantum Access Max with H-ESI II probe
Instrument Type:	triple quadrupole
MS Type:	ESI
MS Comments:	For identification and quantitation, a scheduled MRM method was used. All the details I was able to retrieve are in the table attached. Analyses were performed using a HESI probe with a spray voltage of 3800 V, vaporizer temperature of 350°C, sheath gas at 40 arbU, auxiliary gas at 14 arbU, ion sweep gas at 2 arbU, and a capillary temperature of 320°C; the tube lens settings and monitored transitions (MRM) are reported in the table. The simmer offset was not used, collision gas pressure was set to 1.5 mTorr, and the cycle time was 0.27 s. Selected reaction monitoring was carried out in negative ion mode for diketoderivatives of DCA (precursor/product m/z 387.3/387.3, 4–19 min, collision energy 25, tube lens 110; not detected), monoketoderivatives of DCA (389.3/389.3, 4–19 min, collision energy 25, tube lens 110; PubChem IDs 160738 and 3080612), DCA (391.3/391.3, 4–19 min, collision energy 25, tube lens 110; PubChem ID 222528), tDCA (498.3/498.3, 7–13 min, collision energy 25, tube lens 109; PubChem ID 2733768), and gDCA (448.3/448.3, 8.3–13 min, collision energy 25, tube lens 102; PubChem ID 3035026).
Ion Mode:	NEGATIVE
Analysis Protocol File:	MS_details.pdf

MS ID:	MS007315
Analysis ID:	AN007618
Instrument Name:	TSQ Quantum Access Max with H-ESI II probe
Instrument Type:	triple quadrupole
MS Type:	ESI
MS Comments:	For identification and quantitation, a scheduled MRM method was used. All the details I was able to retrieve are in the table attached. Analyses were performed using a HESI probe with a spray voltage of 3800 V, vaporizer temperature of 350°C, sheath gas at 40 arbU, auxiliary gas at 14 arbU, ion sweep gas at 2 arbU, and a capillary temperature of 320°C; the tube lens settings and monitored transitions (MRM) are reported in the table. The simmer offset was not used, collision gas pressure was set to 1.5 mTorr, and the cycle time was 0.27 s. Selected reaction monitoring was carried out in negative ion mode for diketoderivatives of DCA (precursor/product m/z 387.3/387.3, 4–19 min, collision energy 25, tube lens 110; not detected), monoketoderivatives of DCA (389.3/389.3, 4–19 min, collision energy 25, tube lens 110; PubChem IDs 160738 and 3080612), DCA (391.3/391.3, 4–19 min, collision energy 25, tube lens 110; PubChem ID 222528), tDCA (498.3/498.3, 7–13 min, collision energy 25, tube lens 109; PubChem ID 2733768), and gDCA (448.3/448.3, 8.3–13 min, collision energy 25, tube lens 102; PubChem ID 3035026).
Ion Mode:	NEGATIVE
Analysis Protocol File:	MS_details.pdf