Summary of Study ST004548

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	ST004548
Study Title	Extracellular amino acid 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 amino acid profiles, providing an overview of how bile acid stress and coculture influence amino acid metabolism within this two-member community. C. aerofaciens exhibited increased production of citrulline and ornithine in 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. CA: Collinsella aerofaciens; BT: Bacteroides thetaiotaomicron; Co: Coculture of the two species
Institute	Helmholtz Centre for Environmental Research
Last Name	Yan
First Name	Wang
Address	Permoserstraße 15, Leipzig, Sachsen, 04318, Germany
Email	yan.wang@ufz.de
Phone	+49 341 60251532
Submit Date	2026-01-06
Study Comments	Raw LC–MS acquisition files are uploaded for baseline, undiluted, and diluted study samples. 241025_AA_YW_BHI_baseline.wiff contains the batch with BHI blanks used as baseline. 241021_AA_YW_BTnCA_Co_1zu250.wiff and 241018_AA_YW_BTnCA_Co_1zu25.wiff contain the batches with all study samples measured at two dilution levels.
Analysis Type Detail	LC-MS
Release Date	2026-01-21
Release Version	1

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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:	SU004728
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
SA539223	6_BT_DCA	BT	300µM DCA treated	1
SA539224	7_BT_DCA	BT	300µM DCA treated	2
SA539225	8_BT_DCA	BT	300µM DCA treated	3
SA539226	9_BT_DCA	BT	300µM DCA treated	4
SA539227	10_BT_DCA	BT	300µM DCA treated	5
SA539228	1_BT_C	BT	Control	1
SA539229	2_BT_C	BT	Control	2
SA539230	3_BT_C	BT	Control	3
SA539231	4_BT_C	BT	Control	4
SA539232	5_BT_C	BT	Control	5
SA539233	16_CA_DCA	CA	300µM DCA treated	1
SA539234	17_CA_DCA	CA	300µM DCA treated	2
SA539235	18_CA_DCA	CA	300µM DCA treated	3
SA539236	19_CA_DCA	CA	300µM DCA treated	4
SA539237	20_CA_DCA	CA	300µM DCA treated	5
SA539238	11_CA_C	CA	Control	1
SA539239	12_CA_C	CA	Control	2
SA539240	13_CA_C	CA	Control	3
SA539241	14_CA_C	CA	Control	4
SA539242	15_CA_C	CA	Control	5
SA539243	26_Co_DCA	Co	300µM DCA treated	1
SA539244	27_Co_DCA	Co	300µM DCA treated	2
SA539245	28_Co_DCA	Co	300µM DCA treated	3
SA539246	29_Co_DCA	Co	300µM DCA treated	4
SA539247	30_Co_DCA	Co	300µM DCA treated	5
SA539248	21_Co_C	Co	Control	1
SA539249	22_Co_C	Co	Control	2
SA539250	23_Co_C	Co	Control	3
SA539251	24_Co_C	Co	Control	4
SA539252	25_Co_C	Co	Control	5
SA539253	BHI_1_1zu25	NA	Blank	1
SA539254	BHI_2_1zu25	NA	Blank	2
SA539255	BHI_3_1zu25	NA	Blank	3

Showing results 1 to 33 of 33

Showing results 1 to 33 of 33

Collection:

Collection ID:	CO004721
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

Treatment:

Treatment ID:	TR004737
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:

TR004737

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:	SP004734
Sampleprep Summary:	For extracellular amino acid analysis, 10 µL of the culture supernatant was completely dried. The residue was derivatised with 50 µL of phenylisothiocyanate (PITC) solution (5% PITC in ethanol/water/pyridine, 1:1:1, v/v/v) for 25 min at room temperature, then dried again. The resulting residues were resuspended in 10 µL of extraction solution (5 mM ammonium acetate in methanol) by shaking at 1400 rpm for 10 min in a thermomixer (Eppendorf, Hamburg, Germany), and subsequently diluted with a 1:1 mixture of running buffers A and B prior to analysis.

Sampleprep ID:

SP004734

Sampleprep Summary:

For extracellular amino acid analysis, 10 µL of the culture supernatant was completely dried. The residue was derivatised with 50 µL of phenylisothiocyanate (PITC) solution (5% PITC in ethanol/water/pyridine, 1:1:1, v/v/v) for 25 min at room temperature, then dried again. The resulting residues were resuspended in 10 µL of extraction solution (5 mM ammonium acetate in methanol) by shaking at 1400 rpm for 10 min in a thermomixer (Eppendorf, Hamburg, Germany), and subsequently diluted with a 1:1 mixture of running buffers A and B prior to analysis.

Combined analysis:

Analysis ID	AN007639
Chromatography ID	CH005795
MS ID	MS007335
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Agilent Zorbax Eclipse XDB-C18 column (100 x 3.0 mm, 3.5 µm)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	POSITIVE
Units	µM

Chromatography:

Chromatography ID:	CH005795
Instrument Name:	Waters Acquity
Column Name:	Agilent Zorbax Eclipse XDB-C18 column (100 x 3.0 mm, 3.5 µm)
Column Temperature:	50°C
Flow Gradient:	0–0.5 min, 0% B; 0.5–4 min, 0–70% B; 4–5.3 min, 70% B; 5.3–5.4 min, 70–0% B; and 5.4–7.3 min, 0% B
Flow Rate:	0.5 mL/min
Solvent A:	100% Water; 0.2% formic acid
Solvent B:	100% Acetonitrile; 0.2% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS007335
Analysis ID:	AN007639
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	For identification and quantitation, a scheduled MRM method was used, with specific transitions for every metabolite. Data acquisition and peak integration were performed in Analyst® software (Version 1.7.1). The experiment duration was 7.28 min with a cycle time of 0.8 s, corresponding to 546 cycles, and an MRM detection window of 45 s; no scheduled ionization delay was applied. The target scan time per sMRM experiment was set to 0.8 s to ensure sufficient dwell time and sensitivity across all monitored transitions. The ion source was operated with a curtain gas of 40 psi, IonSpray voltage of 5.5 kV, and a source temperature of 225°C. Ion source gas 1 and gas 2 were set to 40 psi and 60 psi, respectively, providing stable spray conditions and efficient desolvation throughout the analytical run.
Ion Mode:	POSITIVE
Analysis Protocol File:	AA_method.pdf