#METABOLOMICS WORKBENCH katherine_heal_20251104_165618 DATATRACK_ID:6626 STUDY_ID:ST004337 ANALYSIS_ID:AN007243 PROJECT_ID:PR002738 VERSION 1 CREATED_ON November 7, 2025, 6:24 pm #PROJECT PR:PROJECT_TITLE Conserved pathway for homarine catabolism in environmental bacteria PR:PROJECT_SUMMARY Homarine (N-methylpicolinic acid) is a ubiquitous marine metabolite produced by PR:PROJECT_SUMMARY phytoplankton and noted for its bioactivity in marine animals, yet its microbial PR:PROJECT_SUMMARY degradation pathways are uncharacterized. Here, we identify a conserved operon PR:PROJECT_SUMMARY (homABCDER) that mediates homarine catabolism in bacteria using comparative PR:PROJECT_SUMMARY transcriptomics, mutagenesis, and targeted knockouts. Phylogenetic and genomic PR:PROJECT_SUMMARY analyses show this operon distributed across abundant bacterial clades, PR:PROJECT_SUMMARY including coastal copiotrophs (e.g., Rhodobacterales) and open-ocean oligotrophs PR:PROJECT_SUMMARY (e.g., SAR11, SAR116). High-resolution mass spectrometry revealed PR:PROJECT_SUMMARY N-methylglutamic acid and glutamic acid as key metabolic products of homarine in PR:PROJECT_SUMMARY both model and natural systems, with N-methylglutamate dehydrogenase catalyzing PR:PROJECT_SUMMARY their conversion. Metatranscriptomics showed responsive and in situ expression PR:PROJECT_SUMMARY of hom genes aligned with homarine availability. These findings uncover the PR:PROJECT_SUMMARY genetic and metabolic basis of homarine degradation, establish its ecological PR:PROJECT_SUMMARY relevance, and highlight homarine as a versatile growth substrate that feeds PR:PROJECT_SUMMARY into central metabolism via glutamic acid in diverse marine bacteria. PR:INSTITUTE University of Washington, School of Oceanography PR:LAST_NAME Heal PR:FIRST_NAME Katherine PR:ADDRESS 1501 NE Boat Street, Marine Science Building, Room G, Seattle PR:EMAIL katherine.heal@pnnl.gov PR:PHONE 612-616-4840 PR:PUBLICATIONS DOI 10.21203/rs.3.rs-7359689/v1 #STUDY ST:STUDY_TITLE Homarine catabolism: Ruegeria pomeroyi DSS-3 comparative metabolomics under ST:STUDY_TITLE homarine and glucose supported growth ST:STUDY_SUMMARY Cultures of Ruegeria pomeroyi DSS-3 were revived from cryostocks onto ½ YTSS ST:STUDY_SUMMARY agar plates and incubated at 30 °C for 6 days. Single colonies were ST:STUDY_SUMMARY inoculated into 11 mL of glucose minimal media (GMM) and grown overnight at ST:STUDY_SUMMARY 30 °C with shaking at 200 rpm. GMM was prepared using a modified L1 minimal ST:STUDY_SUMMARY medium with glucose 12 mM C as the sole carbon source. All cultures were ST:STUDY_SUMMARY maintained in sterile 15 mL assay tubes. Overnight cultures were diluted to an ST:STUDY_SUMMARY optical density of 0.1 at 600 nm (OD₆₀₀) in fresh GMM, incubated for ST:STUDY_SUMMARY 11–12 h, and amended with glucose (4 mM C, 0.8 mM NH4, control), homarine ST:STUDY_SUMMARY (500 nM C, no additional NH4), or glucose + homarine (1 mM C, 2 mM C, ST:STUDY_SUMMARY respectively with 0.8 mM NH4). Homarine additions were staggered across time ST:STUDY_SUMMARY points to ensure consistent incubation durations. At each time point, samples ST:STUDY_SUMMARY were collected for cell counts and particulate metabolites. For cell counts, ST:STUDY_SUMMARY 1 mL of culture was fixed with glutaraldehyde (final concentration 1%) in ST:STUDY_SUMMARY labeled cryovials, held at 4 °C for 20 minutes, and then stored at ST:STUDY_SUMMARY –80 °C. Particulate metabolites were collected by filtering cultures ST:STUDY_SUMMARY through combusted glass fiber filters using a vacuum manifold set to 8 psi; ST:STUDY_SUMMARY filters were wrapped in combusted foil and flash-frozen in liquid nitrogen. The ST:STUDY_SUMMARY experiment cultures were sampled after two hours in biological triplicate, as ST:STUDY_SUMMARY well as the three control conditions: glucose-only controls, glucose plus ST:STUDY_SUMMARY homarine controls (uninoculated), and a filter blank. ST:INSTITUTE University of Washington, School of Oceanography ST:LAST_NAME Heal ST:FIRST_NAME Katherine ST:ADDRESS 1501 NE Boat Street, Marine Science Building, Room G, Seattle ST:EMAIL katherine.heal@pnnl.gov ST:PHONE 612-616-4840 ST:STUDY_COMMENTS Part of project 6620 ST:PUBLICATIONS DOI 10.21203/rs.3.rs-7359689/v1 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Ruegeria pomeroyi SU:TAXONOMY_ID 246200 SU:GENOTYPE_STRAIN DSS-3 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Gluc_A Sample source:Filtered bacterial cells | treatment:Glucose RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Gluc_A.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Gluc_B Sample source:Filtered bacterial cells | treatment:Glucose RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Gluc_B.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Gluc_C Sample source:Filtered bacterial cells | treatment:Glucose RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Gluc_C.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Hom_A Sample source:Filtered bacterial cells | treatment:Homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Hom_A.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Hom_B Sample source:Filtered bacterial cells | treatment:Homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Hom_B.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Hom_C Sample source:Filtered bacterial cells | treatment:Homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Hom_C.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Mix_A Sample source:Filtered bacterial cells | treatment:Glucose + homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Mix_A.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Mix_B Sample source:Filtered bacterial cells | treatment:Glucose + homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Mix_B.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_Mix_C Sample source:Filtered bacterial cells | treatment:Glucose + homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_Mix_C.mzML; Raw_data_type=Experimental Sample SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_DDAneg20 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_DDAneg20.mzML; Raw_data_type=Pooled experimental sample SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_DDAneg35 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_DDAneg35.mzML; Raw_data_type=Pooled experimental sample SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_DDAneg50 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_DDAneg50.mzML; Raw_data_type=Pooled experimental sample SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_Full1 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_Full1.mzML; Raw_data_type=Pooled experimental sample SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_Full2 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_Full2.mzML; Raw_data_type=Pooled experimental sample SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_Full3 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_Full3.mzML; Raw_data_type=Pooled experimental sample SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_Half1 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_Half1.mzML; Raw_data_type=Pooled experimental sample (half strength) SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_Half2 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_Half2.mzML; Raw_data_type=Pooled experimental sample (half strength) SUBJECT_SAMPLE_FACTORS - 240304_Poo_EpicFate_Half3 Sample source:QC | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Poo_EpicFate_Half3.mzML; Raw_data_type=Pooled experimental sample (half strength) SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_BLK_Gluc_C Sample source:Blank | treatment:Glucose RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_BLK_Gluc_C.mzML; Raw_data_type=Media blank for glucose treatment SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_BLK_Hom_A Sample source:Blank | treatment:Homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_BLK_Hom_A.mzML; Raw_data_type=Media blank for homarine treatment SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_BLK_Hom_B Sample source:Blank | treatment:Homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_BLK_Hom_B.mzML; Raw_data_type=Media blank for homarine treatment SUBJECT_SAMPLE_FACTORS - 240304_Smp_WT_BLK_Mix_A Sample source:Blank | treatment:Glucose + homarine RAW_FILE_NAME(raw_file_name)=240304_Smp_WT_BLK_Mix_A.mzML; Raw_data_type=Media blank for glucose+homarine treatment SUBJECT_SAMPLE_FACTORS - 240304_Std_4uMStdsMix1InH2O_1 Sample source:Standards | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Std_4uMStdsMix1InH2O_1.mzML; Raw_data_type=Standards run SUBJECT_SAMPLE_FACTORS - 240304_Std_4uMStdsMix1InH2O_2 Sample source:Standards | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Std_4uMStdsMix1InH2O_2.mzML; Raw_data_type=Standards run SUBJECT_SAMPLE_FACTORS - 240304_Std_4uMStdsMix2InH2O_1 Sample source:Standards | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Std_4uMStdsMix2InH2O_1.mzML; Raw_data_type=Standards run SUBJECT_SAMPLE_FACTORS - 240304_Std_4uMStdsMix2InH2O_2 Sample source:Standards | treatment:N/A RAW_FILE_NAME(raw_file_name)=240304_Std_4uMStdsMix2InH2O_2.mzML; Raw_data_type=Standards run #COLLECTION CO:COLLECTION_SUMMARY Cultures of Ruegeria pomeroyi DSS-3 were revived from cryostocks onto ½ YTSS CO:COLLECTION_SUMMARY agar plates and incubated at 30 °C for 6 days. Single colonies were CO:COLLECTION_SUMMARY inoculated into 11 mL of glucose minimal media (GMM) and grown overnight at CO:COLLECTION_SUMMARY 30 °C with shaking at 200 rpm. GMM was prepared using a modified L1 minimal CO:COLLECTION_SUMMARY medium with glucose 12 mM C as the sole carbon source. All cultures were CO:COLLECTION_SUMMARY maintained in sterile 15 mL assay tubes. Overnight cultures were diluted to an CO:COLLECTION_SUMMARY optical density of 0.1 at 600 nm (OD₆₀₀) in fresh GMM, incubated for CO:COLLECTION_SUMMARY 11–12 h, and amended with glucose (4 mM C, 0.8 mM NH4, control), homarine CO:COLLECTION_SUMMARY (500 nM C, no additional NH4), or glucose + homarine (1 mM C, 2 mM C, CO:COLLECTION_SUMMARY respectively with 0.8 mM NH4). Homarine additions were staggered across time CO:COLLECTION_SUMMARY points to ensure consistent incubation durations. At each time point, samples CO:COLLECTION_SUMMARY were collected for cell counts and particulate metabolites. For cell counts, CO:COLLECTION_SUMMARY 1 mL of culture was fixed with glutaraldehyde (final concentration 1%) in CO:COLLECTION_SUMMARY labeled cryovials, held at 4 °C for 20 minutes, and then stored at CO:COLLECTION_SUMMARY –80 °C. Particulate metabolites were collected by filtering cultures CO:COLLECTION_SUMMARY through combusted glass fiber filters using a vacuum manifold set to 8 psi; CO:COLLECTION_SUMMARY filters were wrapped in combusted foil and flash-frozen in liquid nitrogen. The CO:COLLECTION_SUMMARY experiment cultures were sampled after two hours in biological triplicate, as CO:COLLECTION_SUMMARY well as the three control conditions: glucose-only controls, glucose plus CO:COLLECTION_SUMMARY homarine controls (uninoculated), and a filter blank. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY Cultures were amended with glucose (4 mM C, 0.8 mM NH4, control), homarine (500 TR:TREATMENT_SUMMARY nM C, no additional NH4), or glucose + homarine (1 mM C, 2 mM C, respectively TR:TREATMENT_SUMMARY with 0.8 mM NH4). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For metabolite extractions, a one phase extraction was performed with SP:SAMPLEPREP_SUMMARY 40:40:20:0.01 methanol:acetonitrile:water:formic acid solution as the extraction SP:SAMPLEPREP_SUMMARY solvent (Canelas et al 2009). Filters were placed in 15 mL Teflon tubes with SP:SAMPLEPREP_SUMMARY pre-chilled extraction solvent, incubated at -20 ºC for 10 minutes, bead beaten SP:SAMPLEPREP_SUMMARY with silica beads, and centrifuged. The solvent was then collected, transferred SP:SAMPLEPREP_SUMMARY into glass tubes, and the procedure was repeated three times while keeping SP:SAMPLEPREP_SUMMARY samples cold as much as possible. Samples were dried down under nitrogen gas, SP:SAMPLEPREP_SUMMARY reconstituted in 400 uL of H2O, and stored at -80 ºC until analysis by LC-MS. SP:SAMPLEPREP_SUMMARY Isotopically-labeled internal standards were added for normalization purposes, SP:SAMPLEPREP_SUMMARY as reported in Table S17 of https://doi.org/10.21203/rs.3.rs-7359689/v1 #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For HILIC chromatography, a SeQuant ZIC-pHILIC column (5 um particle size, 2.1 CH:CHROMATOGRAPHY_SUMMARY mm x 150 mm, from Millipore) was used with 10 mM ammonium carbonate in 85:15 CH:CHROMATOGRAPHY_SUMMARY acetonitrile to water (Solvent A) and 10 mM ammonium carbonate in 85:15 water to CH:CHROMATOGRAPHY_SUMMARY acetonitrile (Solvent B) at a flow rate of 0.15 mL/min. This column was compared CH:CHROMATOGRAPHY_SUMMARY with a Waters UPLC BEH amide and a Millipore cHILIC column; the pHILIC showed CH:CHROMATOGRAPHY_SUMMARY superior reproducibility and peak shapes. The column was held at 100% A for 2 CH:CHROMATOGRAPHY_SUMMARY minutes, ramped to 64% B over 18 minutes, ramped to 100% B over 1 minute, held CH:CHROMATOGRAPHY_SUMMARY at 100% B for 5 minutes, and equilibrated at 100% A for 25 minutes (50 minutes CH:CHROMATOGRAPHY_SUMMARY total). The column was maintained at 30 C. The injection volume was 2 µL for CH:CHROMATOGRAPHY_SUMMARY samples and standard mixes. When starting a batch, the column was equilibrated CH:CHROMATOGRAPHY_SUMMARY at the starting conditions for at least 30 minutes. To improve the performance CH:CHROMATOGRAPHY_SUMMARY of the HILIC column, we maintained the same injection volume, kept the CH:CHROMATOGRAPHY_SUMMARY instrument running water blanks between samples as necessary, and injected CH:CHROMATOGRAPHY_SUMMARY standards in a representative matrix in addition to standards in water. After CH:CHROMATOGRAPHY_SUMMARY each batch, the column was flushed with 10 mM ammonium carbonate in 85:15 water CH:CHROMATOGRAPHY_SUMMARY to acetonitrile for 20 to 30 minutes. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A 85% acetonitrile/15% water; 10 mM ammonium carbonate CH:SOLVENT_B 15% acetonitrile/85% water; 10 mM ammonium carbonate CH:FLOW_GRADIENT The column was held at 100% A for 2 minutes, ramped to 64% B over 18 minutes, CH:FLOW_GRADIENT ramped to 100% B over 1 minute, held at 100% B for 5 minutes, and equilibrated CH:FLOW_GRADIENT at 100% A for 25 minutes CH:FLOW_RATE 0.15 mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS acquisition Comments: Polarity switching was used with a scan range of 60 to MS:MS_COMMENTS 900 m/z and a resolution of 60,000. MS parameters were as follows: capillary MS:MS_COMMENTS temperature was 320 ∞C, the H-ESI spray voltage was 3.3 kV, and the auxiliary MS:MS_COMMENTS gas heater temperature was 100 ∞C. The S-lens RF level was 65. Sheath gas, MS:MS_COMMENTS auxiliary gas, and sweep gas flow rates were maintained at 16, 3, and 1, MS:MS_COMMENTS respectively. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS normalized peak area MS_METABOLITE_DATA_START Samples 240304_Smp_WT_Gluc_A 240304_Smp_WT_Gluc_B 240304_Smp_WT_Gluc_C 240304_Smp_WT_Hom_A 240304_Smp_WT_Hom_B 240304_Smp_WT_Hom_C 240304_Smp_WT_Mix_A 240304_Smp_WT_Mix_B 240304_Smp_WT_Mix_C Factors Sample source:Filtered bacterial cells | treatment:Glucose Sample source:Filtered bacterial cells | treatment:Glucose Sample source:Filtered bacterial cells | treatment:Glucose Sample source:Filtered bacterial cells | treatment:Homarine Sample source:Filtered bacterial cells | treatment:Homarine Sample source:Filtered bacterial cells | treatment:Homarine Sample source:Filtered bacterial cells | treatment:Glucose + homarine Sample source:Filtered bacterial cells | treatment:Glucose + homarine Sample source:Filtered bacterial cells | treatment:Glucose + homarine L-Cysteic acid 1222988.4760399277 1171383.0110978135 1353320.3470441771 4369228.534 12777411.396116065 5854030.664546611 767500.2625711982 390502.36581643927 813594.9958990235 UDP-N-acetylglucosamine 557591.1725464228 260883.80237188455 71551.04796 236973.9825 778880.0513875731 463990.8716432163 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name rt mz L-Cysteic acid 12.15 167.9967 UDP-N-acetylglucosamine 12.75 606.0738 METABOLITES_END #END