Summary of Study ST004337

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 PR002738. The data can be accessed directly via it's Project DOI: 10.21228/M8R54Z This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004337
Study Title	Homarine catabolism: Ruegeria pomeroyi DSS-3 comparative metabolomics under homarine and glucose supported growth
Study Summary	Cultures of Ruegeria pomeroyi DSS-3 were revived from cryostocks onto ½ YTSS agar plates and incubated at 30 °C for 6 days. Single colonies were inoculated into 11 mL of glucose minimal media (GMM) and grown overnight at 30 °C with shaking at 200 rpm. GMM was prepared using a modified L1 minimal medium with glucose 12 mM C as the sole carbon source. All cultures were maintained in sterile 15 mL assay tubes. Overnight cultures were diluted to an optical density of 0.1 at 600 nm (OD₆₀₀) in fresh GMM, incubated for 11–12 h, and amended with glucose (4 mM C, 0.8 mM NH4, control), homarine (500 nM C, no additional NH4), or glucose + homarine (1 mM C, 2 mM C, respectively with 0.8 mM NH4). Homarine additions were staggered across time points to ensure consistent incubation durations. At each time point, samples were collected for cell counts and particulate metabolites. For cell counts, 1 mL of culture was fixed with glutaraldehyde (final concentration 1%) in labeled cryovials, held at 4 °C for 20 minutes, and then stored at –80 °C. Particulate metabolites were collected by filtering cultures through combusted glass fiber filters using a vacuum manifold set to 8 psi; filters were wrapped in combusted foil and flash-frozen in liquid nitrogen. The experiment cultures were sampled after two hours in biological triplicate, as well as the three control conditions: glucose-only controls, glucose plus homarine controls (uninoculated), and a filter blank.
Institute	University of Washington, School of Oceanography
Last Name	Heal
First Name	Katherine
Address	1501 NE Boat Street, Marine Science Building, Room G, Seattle
Email	katherine.heal@pnnl.gov
Phone	612-616-4840
Submit Date	2025-11-04
Study Comments	Part of project 6620
Publications	DOI 10.21203/rs.3.rs-7359689/v1
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2026-01-02
Release Version	1

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Project:

Project ID:	PR002738
Project DOI:	doi: 10.21228/M8R54Z
Project Title:	Conserved pathway for homarine catabolism in environmental bacteria
Project Summary:	Homarine (N-methylpicolinic acid) is a ubiquitous marine metabolite produced by phytoplankton and noted for its bioactivity in marine animals, yet its microbial degradation pathways are uncharacterized. Here, we identify a conserved operon (homABCDER) that mediates homarine catabolism in bacteria using comparative transcriptomics, mutagenesis, and targeted knockouts. Phylogenetic and genomic analyses show this operon distributed across abundant bacterial clades, including coastal copiotrophs (e.g., Rhodobacterales) and open-ocean oligotrophs (e.g., SAR11, SAR116). High-resolution mass spectrometry revealed N-methylglutamic acid and glutamic acid as key metabolic products of homarine in both model and natural systems, with N-methylglutamate dehydrogenase catalyzing their conversion. Metatranscriptomics showed responsive and in situ expression of hom genes aligned with homarine availability. These findings uncover the genetic and metabolic basis of homarine degradation, establish its ecological relevance, and highlight homarine as a versatile growth substrate that feeds into central metabolism via glutamic acid in diverse marine bacteria.
Institute:	University of Washington, School of Oceanography
Last Name:	Heal
First Name:	Katherine
Address:	1501 NE Boat Street, Marine Science Building, Room G, Seattle
Email:	katherine.heal@pnnl.gov
Phone:	612-616-4840

Subject:

Subject ID:	SU004496
Subject Type:	Bacteria
Subject Species:	Ruegeria pomeroyi
Taxonomy ID:	246200
Genotype Strain:	DSS-3

Factors:

Subject type: Bacteria; Subject species: Ruegeria pomeroyi (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	treatment
SA509293	240304_Smp_WT_BLK_Gluc_C	Blank	Glucose
SA509294	240304_Smp_WT_BLK_Mix_A	Blank	Glucose + homarine
SA509295	240304_Smp_WT_BLK_Hom_B	Blank	Homarine
SA509296	240304_Smp_WT_BLK_Hom_A	Blank	Homarine
SA509297	240304_Smp_WT_Gluc_B	Filtered bacterial cells	Glucose
SA509298	240304_Smp_WT_Gluc_A	Filtered bacterial cells	Glucose
SA509299	240304_Smp_WT_Gluc_C	Filtered bacterial cells	Glucose
SA509300	240304_Smp_WT_Mix_B	Filtered bacterial cells	Glucose + homarine
SA509301	240304_Smp_WT_Mix_A	Filtered bacterial cells	Glucose + homarine
SA509302	240304_Smp_WT_Mix_C	Filtered bacterial cells	Glucose + homarine
SA509303	240304_Smp_WT_Hom_A	Filtered bacterial cells	Homarine
SA509304	240304_Smp_WT_Hom_B	Filtered bacterial cells	Homarine
SA509305	240304_Smp_WT_Hom_C	Filtered bacterial cells	Homarine
SA509306	240304_Poo_EpicFate_Half2	QC	N/A
SA509307	240304_Poo_EpicFate_Half3	QC	N/A
SA509308	240304_Poo_EpicFate_Half1	QC	N/A
SA509309	240304_Poo_EpicFate_Full3	QC	N/A
SA509310	240304_Poo_EpicFate_Full1	QC	N/A
SA509311	240304_Poo_EpicFate_DDAneg50	QC	N/A
SA509312	240304_Poo_EpicFate_DDAneg35	QC	N/A
SA509313	240304_Poo_EpicFate_DDAneg20	QC	N/A
SA509314	240304_Poo_EpicFate_Full2	QC	N/A
SA509315	240304_Std_4uMStdsMix1InH2O_1	Standards	N/A
SA509316	240304_Std_4uMStdsMix1InH2O_2	Standards	N/A
SA509317	240304_Std_4uMStdsMix2InH2O_1	Standards	N/A
SA509318	240304_Std_4uMStdsMix2InH2O_2	Standards	N/A

Showing results 1 to 26 of 26

Showing results 1 to 26 of 26

Collection:

Collection ID:	CO004489
Collection Summary:	Cultures of Ruegeria pomeroyi DSS-3 were revived from cryostocks onto ½ YTSS agar plates and incubated at 30 °C for 6 days. Single colonies were inoculated into 11 mL of glucose minimal media (GMM) and grown overnight at 30 °C with shaking at 200 rpm. GMM was prepared using a modified L1 minimal medium with glucose 12 mM C as the sole carbon source. All cultures were maintained in sterile 15 mL assay tubes. Overnight cultures were diluted to an optical density of 0.1 at 600 nm (OD₆₀₀) in fresh GMM, incubated for 11–12 h, and amended with glucose (4 mM C, 0.8 mM NH4, control), homarine (500 nM C, no additional NH4), or glucose + homarine (1 mM C, 2 mM C, respectively with 0.8 mM NH4). Homarine additions were staggered across time points to ensure consistent incubation durations. At each time point, samples were collected for cell counts and particulate metabolites. For cell counts, 1 mL of culture was fixed with glutaraldehyde (final concentration 1%) in labeled cryovials, held at 4 °C for 20 minutes, and then stored at –80 °C. Particulate metabolites were collected by filtering cultures through combusted glass fiber filters using a vacuum manifold set to 8 psi; filters were wrapped in combusted foil and flash-frozen in liquid nitrogen. The experiment cultures were sampled after two hours in biological triplicate, as well as the three control conditions: glucose-only controls, glucose plus homarine controls (uninoculated), and a filter blank.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR004505
Treatment Summary:	Cultures were amended with glucose (4 mM C, 0.8 mM NH4, control), homarine (500 nM C, no additional NH4), or glucose + homarine (1 mM C, 2 mM C, respectively with 0.8 mM NH4).

Sample Preparation:

Sampleprep ID:	SP004502
Sampleprep Summary:	For metabolite extractions, a one phase extraction was performed with 40:40:20:0.01 methanol:acetonitrile:water:formic acid solution as the extraction solvent (Canelas et al 2009). Filters were placed in 15 mL Teflon tubes with pre-chilled extraction solvent, incubated at -20 ºC for 10 minutes, bead beaten with silica beads, and centrifuged. The solvent was then collected, transferred into glass tubes, and the procedure was repeated three times while keeping samples cold as much as possible. Samples were dried down under nitrogen gas, reconstituted in 400 uL of H2O, and stored at -80 ºC until analysis by LC-MS. Isotopically-labeled internal standards were added for normalization purposes, as reported in Table S17 of https://doi.org/10.21203/rs.3.rs-7359689/v1

Sampleprep ID:

SP004502

Sampleprep Summary:

For metabolite extractions, a one phase extraction was performed with 40:40:20:0.01 methanol:acetonitrile:water:formic acid solution as the extraction solvent (Canelas et al 2009). Filters were placed in 15 mL Teflon tubes with pre-chilled extraction solvent, incubated at -20 ºC for 10 minutes, bead beaten with silica beads, and centrifuged. The solvent was then collected, transferred into glass tubes, and the procedure was repeated three times while keeping samples cold as much as possible. Samples were dried down under nitrogen gas, reconstituted in 400 uL of H2O, and stored at -80 ºC until analysis by LC-MS. Isotopically-labeled internal standards were added for normalization purposes, as reported in Table S17 of https://doi.org/10.21203/rs.3.rs-7359689/v1

Combined analysis:

Analysis ID	AN007242	AN007243
Chromatography ID	CH005498	CH005498
MS ID	MS006936	MS006937
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Waters Acquity I-Class	Waters Acquity I-Class
Column	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF-X Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	normalized peak area	normalized peak area

Chromatography:

Chromatography ID:	CH005498
Chromatography Summary:	For HILIC chromatography, a SeQuant ZIC-pHILIC column (5 um particle size, 2.1 mm x 150 mm, from Millipore) was used with 10 mM ammonium carbonate in 85:15 acetonitrile to water (Solvent A) and 10 mM ammonium carbonate in 85:15 water to acetonitrile (Solvent B) at a flow rate of 0.15 mL/min. This column was compared with a Waters UPLC BEH amide and a Millipore cHILIC column; the pHILIC showed superior reproducibility and peak shapes. The column was held at 100% A for 2 minutes, ramped to 64% B over 18 minutes, ramped to 100% B over 1 minute, held at 100% B for 5 minutes, and equilibrated at 100% A for 25 minutes (50 minutes total). The column was maintained at 30 C. The injection volume was 2 µL for samples and standard mixes. When starting a batch, the column was equilibrated at the starting conditions for at least 30 minutes. To improve the performance of the HILIC column, we maintained the same injection volume, kept the instrument running water blanks between samples as necessary, and injected standards in a representative matrix in addition to standards in water. After each batch, the column was flushed with 10 mM ammonium carbonate in 85:15 water to acetonitrile for 20 to 30 minutes.
Instrument Name:	Waters Acquity I-Class
Column Name:	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
Column Temperature:	30
Flow Gradient:	The column was held at 100% A for 2 minutes, ramped to 64% B over 18 minutes, ramped to 100% B over 1 minute, held at 100% B for 5 minutes, and equilibrated at 100% A for 25 minutes
Flow Rate:	0.15 mL/min
Solvent A:	85% acetonitrile/15% water; 10 mM ammonium carbonate
Solvent B:	15% acetonitrile/85% water; 10 mM ammonium carbonate
Chromatography Type:	HILIC

MS:

MS ID:	MS006936
Analysis ID:	AN007242
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS acquisition Comments: Polarity switching was used with a scan range of 60 to 900 m/z and a resolution of 60,000. MS parameters were as follows: capillary temperature was 320 ∞C, the H-ESI spray voltage was 3.3 kV, and the auxiliary gas heater temperature was 100 ∞C. The S-lens RF level was 65. Sheath gas, auxiliary gas, and sweep gas flow rates were maintained at 16, 3, and 1, respectively.
Ion Mode:	POSITIVE

MS ID:	MS006937
Analysis ID:	AN007243
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS acquisition Comments: Polarity switching was used with a scan range of 60 to 900 m/z and a resolution of 60,000. MS parameters were as follows: capillary temperature was 320 ∞C, the H-ESI spray voltage was 3.3 kV, and the auxiliary gas heater temperature was 100 ∞C. The S-lens RF level was 65. Sheath gas, auxiliary gas, and sweep gas flow rates were maintained at 16, 3, and 1, respectively.
Ion Mode:	NEGATIVE