Summary of Study ST004353

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	ST004353
Study Title	Homarine catabolism: Marine microbial isotope-tracing metabolomics experiments for cruise TN412 in the Winter of 2023 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 13C715N-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	2025-11-07
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:	SU004512
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	lon
SA517269	230717_Smp_G5_Hom1_C_B	Particulate	Control	0	19.3316	-138.71
SA517270	230717_Smp_G5_Hom1_C_A	Particulate	Control	0	19.3316	-138.71
SA517271	230717_Smp_G5_Hom1_C_C	Particulate	Control	0	19.3316	-138.71
SA517272	230717_Smp_G5_Hom2_C_C	Particulate	Control	0	2.6222	-140
SA517273	230717_Smp_G5_Hom2_C_B	Particulate	Control	0	2.6222	-140
SA517274	230717_Smp_G5_Hom2_C_A	Particulate	Control	0	2.6222	-140
SA517275	230717_Smp_G5_Hom1_T0_A	Particulate	Homarine (13C7,15N-labelled)	0	19.3316	-138.71
SA517276	230717_Smp_G5_Hom1_T0_C	Particulate	Homarine (13C7,15N-labelled)	0	19.3316	-138.71
SA517277	230717_Smp_G5_Hom1_T0_B	Particulate	Homarine (13C7,15N-labelled)	0	19.3316	-138.71
SA517278	230717_Smp_G5_Hom2_T0_A	Particulate	Homarine (13C7,15N-labelled)	0	2.6222	-140
SA517279	230717_Smp_G5_Hom2_T0_C	Particulate	Homarine (13C7,15N-labelled)	0	2.6222	-140
SA517280	230717_Smp_G5_Hom2_T0_B	Particulate	Homarine (13C7,15N-labelled)	0	2.6222	-140
SA517281	230717_Smp_G5_Hom1_T12_A	Particulate	Homarine (13C7,15N-labelled)	12	19.3316	-138.71
SA517282	230717_Smp_G5_Hom1_T12_B	Particulate	Homarine (13C7,15N-labelled)	12	19.3316	-138.71
SA517283	230717_Smp_G5_Hom1_T12_C	Particulate	Homarine (13C7,15N-labelled)	12	19.3316	-138.71
SA517284	230717_Smp_G5_Hom2_T12_C	Particulate	Homarine (13C7,15N-labelled)	12	2.6222	-140
SA517285	230717_Smp_G5_Hom2_T12_B	Particulate	Homarine (13C7,15N-labelled)	12	2.6222	-140
SA517286	230717_Smp_G5_Hom2_T12_A	Particulate	Homarine (13C7,15N-labelled)	12	2.6222	-140
SA517287	230717_Smp_G5_Hom1_T24_A	Particulate	Homarine (13C7,15N-labelled)	24	19.3316	-138.71
SA517288	230717_Smp_G5_Hom1_T24_C	Particulate	Homarine (13C7,15N-labelled)	24	19.3316	-138.71
SA517289	230717_Smp_G5_Hom1_T24_B	Particulate	Homarine (13C7,15N-labelled)	24	19.3316	-138.71
SA517290	230717_Smp_G5_Hom2_T24_C	Particulate	Homarine (13C7,15N-labelled)	24	2.6222	-140
SA517291	230717_Smp_G5_Hom2_T24_A	Particulate	Homarine (13C7,15N-labelled)	24	2.6222	-140
SA517292	230717_Smp_G5_Hom2_T24_B	Particulate	Homarine (13C7,15N-labelled)	24	2.6222	-140
SA517293	230717_Smp_G5_Hom1_T48_C	Particulate	Homarine (13C7,15N-labelled)	48	19.3316	-138.71
SA517294	230717_Smp_G5_Hom1_T48_B	Particulate	Homarine (13C7,15N-labelled)	48	19.3316	-138.71
SA517295	230717_Smp_G5_Hom1_T48_A	Particulate	Homarine (13C7,15N-labelled)	48	19.3316	-138.71
SA517296	230717_Smp_G5_Hom2_T48_A	Particulate	Homarine (13C7,15N-labelled)	48	2.6222	-140
SA517297	230717_Smp_G5_Hom2_T48_C	Particulate	Homarine (13C7,15N-labelled)	48	2.6222	-140
SA517298	230717_Smp_G5_Hom2_T48_B	Particulate	Homarine (13C7,15N-labelled)	48	2.6222	-140
SA517299	230717_Smp_G5_Hom1_T6_C	Particulate	Homarine (13C7,15N-labelled)	6	19.3316	-138.71
SA517300	230717_Smp_G5_Hom1_T6_B	Particulate	Homarine (13C7,15N-labelled)	6	19.3316	-138.71
SA517301	230717_Smp_G5_Hom1_T6_A	Particulate	Homarine (13C7,15N-labelled)	6	19.3316	-138.71
SA517302	230717_Smp_G5_Hom2_T6_A	Particulate	Homarine (13C7,15N-labelled)	6	2.6222	-140
SA517303	230717_Smp_G5_Hom2_T6_B	Particulate	Homarine (13C7,15N-labelled)	6	2.6222	-140
SA517304	230717_Smp_G5_Hom2_T6_C	Particulate	Homarine (13C7,15N-labelled)	6	2.6222	-140
SA517305	230717_Smp_G5_Hom1_T96_B	Particulate	Homarine (13C7,15N-labelled)	96	19.3316	-138.71
SA517306	230717_Smp_G5_Hom1_T96_C	Particulate	Homarine (13C7,15N-labelled)	96	19.3316	-138.71
SA517307	230717_Smp_G5_Hom1_T96_A	Particulate	Homarine (13C7,15N-labelled)	96	19.3316	-138.71
SA517308	230717_Smp_G5_Hom2_T96_A	Particulate	Homarine (13C7,15N-labelled)	96	2.6222	-140
SA517309	230717_Smp_G5_Hom2_T96_C	Particulate	Homarine (13C7,15N-labelled)	96	2.6222	-140
SA517310	230717_Smp_G5_Hom2_T96_B	Particulate	Homarine (13C7,15N-labelled)	96	2.6222	-140
SA517311	230717_Poo_G5-HomFate2_Full2	QC	N/A	N/A	N/A	N/A
SA517312	230717_Poo_G5-HomFate2_Full7	QC	N/A	N/A	N/A	N/A
SA517313	230717_Poo_G5-HomFate2_Full5	QC	N/A	N/A	N/A	N/A
SA517314	230717_Poo_G5-HomFate2_Full4	QC	N/A	N/A	N/A	N/A
SA517315	230717_Poo_G5-HomFate2_Full3	QC	N/A	N/A	N/A	N/A
SA517316	230717_Poo_G5-HomFate2_Full6	QC	N/A	N/A	N/A	N/A
SA517317	230717_Poo_G5-HomFate2_Full1	QC	N/A	N/A	N/A	N/A
SA517318	230717_Poo_G5-HomFate1_Full2	QC	N/A	N/A	N/A	N/A
SA517319	230717_Poo_G5-HomFate1_Full7	QC	N/A	N/A	N/A	N/A
SA517320	230717_Poo_G5-HomFate1_Full1	QC	N/A	N/A	N/A	N/A
SA517321	230717_Poo_G5-HomFate1_Full6	QC	N/A	N/A	N/A	N/A
SA517322	230717_Poo_G5-HomFate1_Full3	QC	N/A	N/A	N/A	N/A
SA517323	230717_Poo_G5-HomFate1_Full4	QC	N/A	N/A	N/A	N/A
SA517324	230717_Poo_G5-HomFate1_Full5	QC	N/A	N/A	N/A	N/A
SA517325	230717_Std_4uMStdsMix1InH2O_1	Standards	N/A	N/A	N/A	N/A
SA517326	230717_Std_4uMStdsMix1InH2O_2	Standards	N/A	N/A	N/A	N/A
SA517327	230717_Std_4uMStdsMix1InH2O_3	Standards	N/A	N/A	N/A	N/A
SA517328	230717_Std_4uMStdsMix1InH2O_5	Standards	N/A	N/A	N/A	N/A
SA517329	230717_Std_4uMStdsMix1InH2O_6	Standards	N/A	N/A	N/A	N/A
SA517330	230717_Std_4uMStdsMix2InH2O_1	Standards	N/A	N/A	N/A	N/A
SA517331	230717_Std_4uMStdsMix2InH2O_2	Standards	N/A	N/A	N/A	N/A
SA517332	230717_Std_4uMStdsMix2InH2O_3	Standards	N/A	N/A	N/A	N/A
SA517333	230717_Std_4uMStdsMix2InH2O_4	Standards	N/A	N/A	N/A	N/A
SA517334	230717_Std_4uMStdsMix2InH2O_5	Standards	N/A	N/A	N/A	N/A
SA517335	230717_Std_4uMStdsMix2InH2O_6	Standards	N/A	N/A	N/A	N/A
SA517336	230717_Std_4uMStdsMix1InH2O_4	Standards	N/A	N/A	N/A	N/A

Showing results 1 to 68 of 68

Showing results 1 to 68 of 68

Collection:

Collection ID:	CO004505
Collection Summary:	Experiments using 13C7,15N-homarine-homarine were performed on research cruise TN412 in the Winter of 2023 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). The preparation of the 13C7-15N-labeled homarine is described in detail in 10.21203/rs.3.rs-7359689/v1. Seawater was collected through a trace metal clean stayfish system suspended at a depth of 8 m prefiltered through 100 µm nylon mesh. Triplicate samples were collected into acid washed 2 L polycarbonate bottles, spiked with 90 nM of 13C7-15N-labeled homarine, and incubated in temperature and light-controlled incubators for 5 different timepoints (6, 12, 24, 48 and 96 hours). Triplicates of spiked and unspiked samples were filtered as quickly as possible, no more than 30 minutes (T0, and unamended control samples, respectively). 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:	TR004521
Treatment Summary:	Triplicate samples were collected into acid washed 2 L polycarbonate bottles, spiked with 90 nM of 13C7-15N-labeled homarine, and incubated in temperature and light-controlled incubators for 5 different timepoints (6, 12, 24, 48 and 96 hours). Triplicates of spiked and unspiked samples were filtered as quickly as possible, no more than 30 minutes (T0, and unamended control samples, respectively).

Sample Preparation:

Sampleprep ID:	SP004518
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:

SP004518

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	AN007267	AN007268
Chromatography ID	CH005515	CH005515
MS ID	MS006961	MS006962
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:	CH005515
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:	MS006961
Analysis ID:	AN007267
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:	MS006962
Analysis ID:	AN007268
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