Summary of Study ST004324
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 | ST004324 |
| Study Title | Homarine catabolism: Cobetia sp. OBi1 comparative metabolomics under homarine and glucose supported growth |
| Study Summary | To uncover genes involved in homarine degradation, we grew Cobetia sp. OBi1 in minimal media with homarine as the sole carbon source and conducted comparative transcriptomics and metabolomics experiments. We compared three conditions: glucose (12 mM C, 0.8 mM NH4, control), homarine (12 mM C, no additional NH4), and glucose + homarine (12 mM C, 1 mM C, respectively with 0.8 mM NH4). |
| Institute | University of Washington, School of Oceanography |
| Last Name | Heal |
| First Name | Katherine |
| Address | 1501 NE Boat Street, Marine Science Building, Room G, Seattle |
| katherine.heal@pnnl.gov | |
| Phone | 612-616-4840 |
| Submit Date | 2025-10-27 |
| 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 |
Select appropriate tab below to view additional metadata details:
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: | SU004479 |
| Subject Type: | Bacteria |
| Subject Species: | Cobetia marina |
| Taxonomy ID: | 28258 |
| Genotype Strain: | OBi1 |
Factors:
Subject type: Bacteria; Subject species: Cobetia marina (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment |
|---|---|---|---|
| SA505970 | 211202_Smp_OBi1_2 | Cell pellet | Glucose |
| SA505971 | 211202_Smp_OBi1_1 | Cell pellet | Glucose |
| SA505972 | 211202_Smp_OBi1_3 | Cell pellet | Glucose |
| SA505973 | 211202_Smp_OBi1_7 | Cell pellet | Glucose + homarine |
| SA505974 | 211202_Smp_OBi1_8 | Cell pellet | Glucose + homarine |
| SA505975 | 211202_Smp_OBi1_9 | Cell pellet | Glucose + homarine |
| SA505976 | 211202_Smp_OBi1_4 | Cell pellet | Homarine |
| SA505977 | 211202_Smp_OBi1_5 | Cell pellet | Homarine |
| SA505978 | 211202_Smp_OBi1_6 | Cell pellet | Homarine |
| SA505979 | 220302_Smp_OBi1_2 | Supernatant | Glucose |
| SA505980 | 220302_Smp_OBi1_3 | Supernatant | Glucose |
| SA505981 | 220302_Smp_OBi1_1 | Supernatant | Glucose |
| SA505982 | 220302_Smp_OBi1_7 | Supernatant | Glucose + homarine |
| SA505983 | 220302_Smp_OBi1_8 | Supernatant | Glucose + homarine |
| SA505984 | 220302_Smp_OBi1_9 | Supernatant | Glucose + homarine |
| SA505985 | 220302_Smp_OBi1_4 | Supernatant | Homarine |
| SA505986 | 220302_Smp_OBi1_5 | Supernatant | Homarine |
| SA505987 | 220302_Smp_OBi1_6 | Supernatant | Homarine |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO004472 |
| Collection Summary: | Isolate Owen Beach isolate 1 (OBi1) was obtained by enrichment culturing of seawater collected in Owen Beach (Tacoma, Washington). Seawater was collected in sterile polycarbonate bottles in February 2020. We amended 100 mL seawater samples with 12 mM C homarine and 0.1 mM sodium phosphate and incubated in aerated flasks at 22°C. Culture OD600 was measured daily to monitor growth. After three days, 50 uL samples were streaked on agar plates made with marine broth (Difco 2216) to isolate single colonies. Single colonies were picked and re-streaked twice in the same agar media. The isolated colonies were tested for growth on homarine in seawater from Owen Beach sterilized by filtering through a 0.22 um PES membrane and autoclaving. We supplied the medium with homarine and phosphate as before. OBi1 colonies re-grew on homarine and the isolate was selected for further analysis. Cobetia sp. OBi1 was grown in three conditions, as described for comparative transcriptomic analyses: homarine, glucose, and glucose+homarine. Overnight cultures of Cobetia sp. OBi1 (100 mL) were grown at 25°C grown in glucose-amended seawater media as described for Cobetia sp. OBi1 transcriptomics experiments. Next, 10 mL subsamples were taken from overnight culture and centrifuged for 15 minutes at 2800 g, and resuspended in the experimental growth media homarine, glucose, or glucose+homarine, in triplicate (again, as described for Cobetia sp. OBi1 transcriptomics experiments). These samples were incubated for 1 hr at 25°C in a dark shaker before harvesting. Cells were harvested by centrifugation for 15 minutes at 2800 g, and supernatant was collected and filtered through a 0.22 um PES membrane filter. Cell pellets and supernatant samples were stored at -80°C and -20°C, respectively. |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR004488 |
| Treatment Summary: | Cobetia sp. OBi1 was grown in three conditions, as described for comparative transcriptomic analyses: homarine, glucose, and glucose+homarine. Overnight cultures of Cobetia sp. OBi1 (100 mL) were grown at 25°C grown in glucose-amended seawater media as described for Cobetia sp. OBi1 transcriptomics experiments. |
Sample Preparation:
| Sampleprep ID: | SP004485 |
| 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. 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 | AN007206 | AN007207 |
|---|---|---|
| Chromatography ID | CH005473 | CH005473 |
| MS ID | MS006901 | MS006902 |
| 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 hybrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | normalized peak area | normalized peak area |
Chromatography:
| Chromatography ID: | CH005473 |
| 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. |
| Methods ID: | CH001683 |
| 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: | MS006901 |
| Analysis ID: | AN007206 |
| 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: | MS006902 |
| Analysis ID: | AN007207 |
| 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: | NEGATIVE |
