Summary of Study ST004352

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	ST004352
Study Title	Homarine catabolism: Marine microbial isotope-tracing metabolomics experiments for cruise TN397 in the Fall of 2021 at two different stations in the North Pacific
Study Summary	Stable-isotope probing was performed to track homarine degradation products in natural marine microbial communities from three locations (Figure 2D of DOI 10.21203/rs.3.rs-7359689/v1). Seawater incubated with isotopically-labeled 2H3-homarine was analyzed for the compounds enriched in our model organisms (see other studies within this project), with the isotopic labels.
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	2025-11-06
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:	SU004511
Subject Type:	Water sample

Factors:

Subject type: Water sample; Subject species: - (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	treatment	timepoint	lat_collected	lon_collected	date_collected
SA517195	230724_Smp_G4_2_C-H_t0_A	Filtered Cells	Control	0	0.1812	-141.9465	20211208
SA517196	230724_Smp_G4_2_C-H_t0_C	Filtered Cells	Control	0	0.1812	-141.9465	20211208
SA517197	230724_Smp_G4_2_C-H_t0_B	Filtered Cells	Control	0	0.1812	-141.9465	20211208
SA517198	230724_Smp_G4_1_C-H_t0_B	Filtered Cells	Control	0	27.0673	-126.6739	20211123
SA517199	230724_Smp_G4_1_C-H_t0_A	Filtered Cells	Control	0	27.0673	-126.6739	20211123
SA517200	230724_Smp_G4_1_C-H_t0_C	Filtered Cells	Control	0	27.0673	-126.6739	20211123
SA517201	230724_Smp_G4_2_C-H_t24_A	Filtered Cells	Control	24	0.1812	-141.9465	20211208
SA517202	230724_Smp_G4_2_C-H_t24_B	Filtered Cells	Control	24	0.1812	-141.9465	20211208
SA517203	230724_Smp_G4_2_C-H_t24_C	Filtered Cells	Control	24	0.1812	-141.9465	20211208
SA517204	230724_Smp_G4_1_C-H_t24_A	Filtered Cells	Control	24	27.0673	-126.6739	20211123
SA517205	230724_Smp_G4_1_C-H_t24_C	Filtered Cells	Control	24	27.0673	-126.6739	20211123
SA517206	230724_Smp_G4_1_C-H_t24_B	Filtered Cells	Control	24	27.0673	-126.6739	20211123
SA517207	230724_Smp_G4_2_C-H_t2_B	Filtered Cells	Control	2	0.1812	-141.9465	20211208
SA517208	230724_Smp_G4_2_C-H_t2_A	Filtered Cells	Control	2	0.1812	-141.9465	20211208
SA517209	230724_Smp_G4_2_C-H_t2_C	Filtered Cells	Control	2	0.1812	-141.9465	20211208
SA517210	230724_Smp_G4_1_C-H_t2_B	Filtered Cells	Control	2	27.0673	-126.6739	20211123
SA517211	230724_Smp_G4_1_C-H_t2_C	Filtered Cells	Control	2	27.0673	-126.6739	20211123
SA517212	230724_Smp_G4_1_C-H_t2_A	Filtered Cells	Control	2	27.0673	-126.6739	20211123
SA517213	230724_Smp_G4_2_C-H_t48_B	Filtered Cells	Control	48	0.1812	-141.9465	20211208
SA517214	230724_Smp_G4_2_C-H_t48_A	Filtered Cells	Control	48	0.1812	-141.9465	20211208
SA517215	230724_Smp_G4_2_C-H_t48_C	Filtered Cells	Control	48	0.1812	-141.9465	20211208
SA517216	230724_Smp_G4_1_C-H_t48_C	Filtered Cells	Control	48	27.0673	-126.6739	20211123
SA517217	230724_Smp_G4_1_C-H_t48_B	Filtered Cells	Control	48	27.0673	-126.6739	20211123
SA517218	230724_Smp_G4_1_C-H_t48_A	Filtered Cells	Control	48	27.0673	-126.6739	20211123
SA517219	230724_Smp_G4_2_H_t24_A	Filtered Cells	Homarine (2H3-labelled)	24	0.1812	-141.9465	20211208
SA517220	230724_Smp_G4_2_H_t24_C	Filtered Cells	Homarine (2H3-labelled)	24	0.1812	-141.9465	20211208
SA517221	230724_Smp_G4_2_H_t24_B	Filtered Cells	Homarine (2H3-labelled)	24	0.1812	-141.9465	20211208
SA517222	230724_Smp_G4_1_H_t24_C	Filtered Cells	Homarine (2H3-labelled)	24	27.0673	-126.6739	20211123
SA517223	230724_Smp_G4_1_H_t24_B	Filtered Cells	Homarine (2H3-labelled)	24	27.0673	-126.6739	20211123
SA517224	230724_Smp_G4_1_H_t24_A	Filtered Cells	Homarine (2H3-labelled)	24	27.0673	-126.6739	20211123
SA517225	230724_Smp_G4_2_H_t2_A	Filtered Cells	Homarine (2H3-labelled)	2	0.1812	-141.9465	20211208
SA517226	230724_Smp_G4_2_H_t2_B	Filtered Cells	Homarine (2H3-labelled)	2	0.1812	-141.9465	20211208
SA517227	230724_Smp_G4_2_H_t2_C	Filtered Cells	Homarine (2H3-labelled)	2	0.1812	-141.9465	20211208
SA517228	230724_Smp_G4_1_H_t2_C	Filtered Cells	Homarine (2H3-labelled)	2	27.0673	-126.6739	20211123
SA517229	230724_Smp_G4_1_H_t2_B	Filtered Cells	Homarine (2H3-labelled)	2	27.0673	-126.6739	20211123
SA517230	230724_Smp_G4_1_H_t2_A	Filtered Cells	Homarine (2H3-labelled)	2	27.0673	-126.6739	20211123
SA517231	230724_Smp_G4_2_H_t48_B	Filtered Cells	Homarine (2H3-labelled)	48	0.1812	-141.9465	20211208
SA517232	230724_Smp_G4_2_H_t48_C	Filtered Cells	Homarine (2H3-labelled)	48	0.1812	-141.9465	20211208
SA517233	230724_Smp_G4_2_H_t48_A	Filtered Cells	Homarine (2H3-labelled)	48	0.1812	-141.9465	20211208
SA517234	230724_Smp_G4_1_H_t48_B	Filtered Cells	Homarine (2H3-labelled)	48	27.0673	-126.6739	20211123
SA517235	230724_Smp_G4_1_H_t48_C	Filtered Cells	Homarine (2H3-labelled)	48	27.0673	-126.6739	20211123
SA517236	230724_Smp_G4_1_H_t48_A	Filtered Cells	Homarine (2H3-labelled)	48	27.0673	-126.6739	20211123
SA517237	230724_Poo_G4-MethylHomFate_DDAneg20	QC	N/A	N/A	N/A	N/A	N/A
SA517238	230724_Poo_G4-MethylHomFate_Full2	QC	N/A	N/A	N/A	N/A	N/A
SA517239	230724_Poo_G4-MethylHomFate_Full9	QC	N/A	N/A	N/A	N/A	N/A
SA517240	230724_Poo_G4-MethylHomFate_Full8	QC	N/A	N/A	N/A	N/A	N/A
SA517241	230724_Poo_G4-MethylHomFate_Full7	QC	N/A	N/A	N/A	N/A	N/A
SA517242	230724_Poo_G4-MethylHomFate_DDAneg35	QC	N/A	N/A	N/A	N/A	N/A
SA517243	230724_Poo_G4-MethylHomFate_Full5	QC	N/A	N/A	N/A	N/A	N/A
SA517244	230724_Poo_G4-MethylHomFate_Full4	QC	N/A	N/A	N/A	N/A	N/A
SA517245	230724_Poo_G4-MethylHomFate_Full3	QC	N/A	N/A	N/A	N/A	N/A
SA517246	230724_Poo_G4-MethylHomFate_Full6	QC	N/A	N/A	N/A	N/A	N/A
SA517247	230724_Poo_G4-MethylHomFate_Full14	QC	N/A	N/A	N/A	N/A	N/A
SA517248	230724_Poo_G4-MethylHomFate_DDApos35	QC	N/A	N/A	N/A	N/A	N/A
SA517249	230724_Poo_G4-MethylHomFate_Full13	QC	N/A	N/A	N/A	N/A	N/A
SA517250	230724_Poo_G4-MethylHomFate_DDAneg50	QC	N/A	N/A	N/A	N/A	N/A
SA517251	230724_Poo_G4-MethylHomFate_DDApos20	QC	N/A	N/A	N/A	N/A	N/A
SA517252	230724_Poo_G4-MethylHomFate_Full12	QC	N/A	N/A	N/A	N/A	N/A
SA517253	230724_Poo_G4-MethylHomFate_DDApos50	QC	N/A	N/A	N/A	N/A	N/A
SA517254	230724_Poo_G4-MethylHomFate_Full1	QC	N/A	N/A	N/A	N/A	N/A
SA517255	230724_Poo_G4-MethylHomFate_Full10	QC	N/A	N/A	N/A	N/A	N/A
SA517256	230724_Poo_G4-MethylHomFate_Full11	QC	N/A	N/A	N/A	N/A	N/A
SA517257	230724_Std_4uMStdsMix1InH2O_1	Standards	N/A	N/A	N/A	N/A	N/A
SA517258	230724_Std_4uMStdsMix1InH2O_2	Standards	N/A	N/A	N/A	N/A	N/A
SA517259	230724_Std_4uMStdsMix1InH2O_4	Standards	N/A	N/A	N/A	N/A	N/A
SA517260	230724_Std_4uMStdsMix1InH2O_5	Standards	N/A	N/A	N/A	N/A	N/A
SA517261	230724_Std_4uMStdsMix1InH2O_6	Standards	N/A	N/A	N/A	N/A	N/A
SA517262	230724_Std_4uMStdsMix2InH2O_1	Standards	N/A	N/A	N/A	N/A	N/A
SA517263	230724_Std_4uMStdsMix2InH2O_2	Standards	N/A	N/A	N/A	N/A	N/A
SA517264	230724_Std_4uMStdsMix2InH2O_3	Standards	N/A	N/A	N/A	N/A	N/A
SA517265	230724_Std_4uMStdsMix2InH2O_4	Standards	N/A	N/A	N/A	N/A	N/A
SA517266	230724_Std_4uMStdsMix2InH2O_5	Standards	N/A	N/A	N/A	N/A	N/A
SA517267	230724_Std_4uMStdsMix2InH2O_6	Standards	N/A	N/A	N/A	N/A	N/A
SA517268	230724_Std_4uMStdsMix1InH2O_3	Standards	N/A	N/A	N/A	N/A	N/A

Showing results 1 to 74 of 74

Showing results 1 to 74 of 74

Collection:

Collection ID:	CO004504
Collection Summary:	Experiments using 2H3-homarine were performed on research cruise TN397 in the Fall of 2021 at two different stations in the North Pacific (described in Table S8 and displayed in Figure 2 of 10.21203/rs.3.rs-7359689/v1). 2H3-homarine was purchased from Toronto Research Chemicals and was injected onto a Q-Exactive HF Orbitrap Mass Spectrometer (QE-HF) to confirm the mass of the deuterium label (141.0743 m/z) and the retention time (6.4 minutes, same as non-labeled homarine). Seawater was collected into 21 acid washed 10 L polycarbonate carboys from a trace metal clean stayfish system suspended at a depth of 8 m and prefiltered through 100 µm nylon mesh. Three unamended samples were collected immediately after seawater collection to provide samples of the starting community. Nine treatment bottles were spiked with 500 nM 2H3-homarine with nine control bottles receiving no additions. Bottles were incubated in blue-shaded temperature and light-controlled incubators designed to mimic mixed-layer conditions of the sampling location. Triplicate bottles with and without homarine addition were harvested at 2, 24, and 48 hours. All particulate samples (4 L) were collected using peristaltic pumps onto Durapore® 0.22 μm, 47 mm, hydrophilic PVDF membrane filters, flash frozen in liquid nitrogen, and stored at -80°C.
Sample Type:	Marine particulate matter

Treatment:

Treatment ID:	TR004520
Treatment Summary:	Nine treatment bottles were spiked with 500 nM 2H3-homarine with nine control bottles receiving no additions. Bottles were incubated in blue-shaded temperature and light-controlled incubators designed to mimic mixed-layer conditions of the sampling location. Triplicate bottles with and without homarine addition were harvested at 2, 24, and 48 hours. The experiment was repeated with the same treatments at a second location.

Treatment ID:

TR004520

Treatment Summary:

Nine treatment bottles were spiked with 500 nM 2H3-homarine with nine control bottles receiving no additions. Bottles were incubated in blue-shaded temperature and light-controlled incubators designed to mimic mixed-layer conditions of the sampling location. Triplicate bottles with and without homarine addition were harvested at 2, 24, and 48 hours. The experiment was repeated with the same treatments at a second location.

Sample Preparation:

Sampleprep ID:	SP004517
Sampleprep Summary:	For particulate metabolomics, cell pellets were extracted using a combination of mechanical and chemical disruption techniques as described in previous work (Boysen et al 2018). Metabolites from the supernatant were extracted using a cation-exchange-based solid phase extraction technique as described previously (Sacks et al 2022), with 1 mL of supernatant diluted into 10 mL of HPLC grade water. To prevent confusion using the isotope labels, we used a subset of isotopically-labeled internal standards, as reported in Table S17 of https://doi.org/10.21203/rs.3.rs-7359689/v1.

Sampleprep ID:

SP004517

Sampleprep Summary:

For particulate metabolomics, cell pellets were extracted using a combination of mechanical and chemical disruption techniques as described in previous work (Boysen et al 2018). Metabolites from the supernatant were extracted using a cation-exchange-based solid phase extraction technique as described previously (Sacks et al 2022), with 1 mL of supernatant diluted into 10 mL of HPLC grade water. To prevent confusion using the isotope labels, we used a subset of isotopically-labeled internal standards, as reported in Table S17 of https://doi.org/10.21203/rs.3.rs-7359689/v1.

Combined analysis:

Analysis ID	AN007265	AN007266
Chromatography ID	CH005514	CH005514
MS ID	MS006959	MS006960
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:	CH005514
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:	MS006959
Analysis ID:	AN007265
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:	MS006960
Analysis ID:	AN007266
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