#METABOLOMICS WORKBENCH uchimiya_20201018_133611 DATATRACK_ID:2215 STUDY_ID:ST001512 ANALYSIS_ID:AN002506 VERSION 1 CREATED_ON 07-31-2023 #PROJECT PR:PROJECT_TITLE Diel investments in phytoplankton metabolite production influenced by associated PR:PROJECT_TITLE heterotrophic bacteria PR:INSTITUTE University of Georgia PR:DEPARTMENT Department of Marine Sciences; Complex Carbohydrate Research Center PR:LABORATORY Moran Lab; Edison Lab PR:LAST_NAME Uchimiya PR:FIRST_NAME Mario PR:ADDRESS 315 Riverbend Rd, Athens, GA 30602 PR:EMAIL mario.uchimiya@uga.edu PR:PHONE (706) 542-8387 PR:FUNDING_SOURCE The Gordon and Betty Moore Foundation (5503); NSF (IOS-1656311); The Simons PR:FUNDING_SOURCE Foundation (542391); JSPS (Research Fellowship for Young Scientists and PR:FUNDING_SOURCE Grant-in-Aid for JSPS Fellows); Swedish Research Council (2018-06571) PR:DOI http://dx.doi.org/10.21228/M80408 #STUDY ST:STUDY_TITLE Diel investments in phytoplankton metabolite production influenced by associated ST:STUDY_TITLE heterotrophic bacteria ST:STUDY_TYPE Incubation experiment ST:STUDY_SUMMARY Organic substrate transfer between photoautotrophic and heterotrophic microbes ST:STUDY_SUMMARY in the surface ocean is a central but poorly understood process in the global ST:STUDY_SUMMARY carbon cycle. This study developed a co-culture system of marine diatom ST:STUDY_SUMMARY Thalassiosira pseudonana and heterotrophic bacterium Ruegeria pomeroyi, and ST:STUDY_SUMMARY addressed diel changes in phytoplankton endometabolite production using nuclear ST:STUDY_SUMMARY magnetic resonance (NMR) and bacterial metabolite consumption using gene ST:STUDY_SUMMARY expression. Here we deposit data for NMR analysis from the study. Samples were ST:STUDY_SUMMARY collected every 6 hours over two days under a diel light cycle. During the ST:STUDY_SUMMARY course of the study, we observed an increase in some phytoplankton ST:STUDY_SUMMARY endometabolites presumably due to the effects of the associated bacteria. We ST:STUDY_SUMMARY introduced an additional experiment and tested this possibility by comparing ST:STUDY_SUMMARY phytoplankton endometabolite accumulation between axenic treatments and bacteria ST:STUDY_SUMMARY coculture treatments. ST:INSTITUTE University of Georgia ST:DEPARTMENT Department of Marine Sciences; Complex Carbohydrate Research Center ST:LABORATORY Moran Lab; Edison Lab ST:LAST_NAME Uchimiya ST:FIRST_NAME Mario ST:ADDRESS 315 Riverbend Rd, Athens, GA 30602 ST:EMAIL mario.uchimiya@uga.edu ST:PHONE (706) 542-8387 ST:SUBMIT_DATE 2020-10-18 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Thalassiosira pseudonana CCMP1335 SU:TAXONOMY_ID 296543 SU:GENOTYPE_STRAIN CCMP1335 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 18 Collection time:12 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=18; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Noon-1; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 20 Collection time:12 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=20; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Noon-1; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 22 Collection time:12 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=22; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Noon-1; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 24 Collection time:18 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=24; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-afternoon-1; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 26 Collection time:18 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=26; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-afternoon-1; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 28 Collection time:18 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=28; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-afternoon-1; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 30 Collection time:24.5 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=30; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-2; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 32 Collection time:24.5 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=32; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-2; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 34 Collection time:24.5 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=34; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-2; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 36 Collection time:30.6 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=36; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-morning-2; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 38 Collection time:30.6 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=38; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-morning-2; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 40 Collection time:30.6 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=40; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-morning-2; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 42 Collection time:35.8 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=42; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Noon-2; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 44 Collection time:35.8 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=44; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Noon-2; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 46 Collection time:35.8 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=46; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Noon-2; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 48 Collection time:40.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=48; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-afternoon-2; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 50 Collection time:40.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=50; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-afternoon-2; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 52 Collection time:40.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=52; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-afternoon-2; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 54 Collection time:44.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=54; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-3; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 56 Collection time:44.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=56; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-3; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 58 Collection time:44.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=58; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-3; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 12 Collection time:5.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=12; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-morning-1; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 14 Collection time:5.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=14; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-morning-1; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 16 Collection time:5.9 | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=16; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Mid-morning-1; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 10 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=10; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-1; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 103 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=103; Experiment name=Compound annotation; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-2; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 2 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=2; Experiment name=Compound annotation; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-2; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 3 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=3; Experiment name=Compound annotation; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-2; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 43 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=43; Experiment name=Test of model prediction experiment; Sample descritpion=Sample to check experimental background; Biological replicate=- SUBJECT_SAMPLE_FACTORS - 51 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=51; Experiment name=Test of model prediction experiment; Sample descritpion=Sample to check experimental background; Biological replicate=- SUBJECT_SAMPLE_FACTORS - 57 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=57; Experiment name=Test of model prediction experiment; Sample descritpion=Sample to check experimental background; Biological replicate=- SUBJECT_SAMPLE_FACTORS - 6 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=6; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-1; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 60 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=60; Experiment name=Diel experiment; Sample descritpion=Sample to check experimental background; Biological replicate=- SUBJECT_SAMPLE_FACTORS - 8 Collection time:- | Prediction Model (Coculture vs Axenic):- Sample_Data:RAW_FILE_NAME=8; Experiment name=Diel experiment; Sample descritpion=Phytoplankton-bacteria coculture under diel light cycles, Night-1; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 31 Collection time:- | Prediction Model (Coculture vs Axenic):Axenic Sample_Data:RAW_FILE_NAME=31; Experiment name=Test of model prediction experiment; Sample descritpion=Phytoplankton axenic mono-culture, Day 15; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 33 Collection time:- | Prediction Model (Coculture vs Axenic):Axenic Sample_Data:RAW_FILE_NAME=33; Experiment name=Test of model prediction experiment; Sample descritpion=Phytoplankton axenic mono-culture, Day 15; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 35 Collection time:- | Prediction Model (Coculture vs Axenic):Axenic Sample_Data:RAW_FILE_NAME=35; Experiment name=Test of model prediction experiment; Sample descritpion=Phytoplankton axenic mono-culture, Day 15; Biological replicate=3 SUBJECT_SAMPLE_FACTORS - 25 Collection time:- | Prediction Model (Coculture vs Axenic):Coculture Sample_Data:RAW_FILE_NAME=25; Experiment name=Test of model prediction experiment; Sample descritpion=Phytoplankton-bacteria coculture, Day 15; Biological replicate=1 SUBJECT_SAMPLE_FACTORS - 27 Collection time:- | Prediction Model (Coculture vs Axenic):Coculture Sample_Data:RAW_FILE_NAME=27; Experiment name=Test of model prediction experiment; Sample descritpion=Phytoplankton-bacteria coculture, Day 15; Biological replicate=2 SUBJECT_SAMPLE_FACTORS - 29 Collection time:- | Prediction Model (Coculture vs Axenic):Coculture Sample_Data:RAW_FILE_NAME=29; Experiment name=Test of model prediction experiment; Sample descritpion=Phytoplankton-bacteria coculture, Day 15; Biological replicate=3 #COLLECTION CO:COLLECTION_SUMMARY Diel experiment – Samples were collected every 6 h over 2 days. 500 mL of CO:COLLECTION_SUMMARY diatom cells were collected by filtering the culture onto 2.0-µm-pore-size PCTE CO:COLLECTION_SUMMARY membrane filters (MilliporeSigma Isopore). Filters were kept in 50 mL tubes CO:COLLECTION_SUMMARY (Falcon) and stored at -80oC until processing. Test of model prediction CO:COLLECTION_SUMMARY experiment – The same procedure was used except that samples were collected at CO:COLLECTION_SUMMARY day 15 and 700 mL of the culture was filtered. CO:COLLECTION_PROTOCOL_FILENAME uchimiya_20201018_2_Collection protocol_UGA_phytoplankton_Oct2020.docx CO:COLLECTION_PROTOCOL_COMMENTS Collection summary is in 2_Collection protocol_UGA_phytoplankton_Oct2020.docx CO:SAMPLE_TYPE Algae #TREATMENT TR:TREATMENT_SUMMARY Diel experiment – An axenic strain of marine diatom Thalassiosira pseudonana TR:TREATMENT_SUMMARY CCMP1335 was cultured at 18 oC in three replicate 15-L polycarbonate bottles TR:TREATMENT_SUMMARY containing 10 L of L1 medium in which NaH13CO3 (Cambridge Isotope Laboratories, TR:TREATMENT_SUMMARY CLM-441) was used for sodium bicarbonate. The light cycle consisted of 16 h TR:TREATMENT_SUMMARY light, during which light intensity varied gradually between 0 and 150 µmol TR:TREATMENT_SUMMARY photon m-2 s-1 with a maximum intensity at noon, followed by 8 h of dark. TR:TREATMENT_SUMMARY Bacterial strain Ruegeria pomeroyi DSS-3 was grown at 30oC on ½ YTSS agar and TR:TREATMENT_SUMMARY transferred to ½ YTSS liquid2 medium for overnight growth. Axenic T. pseudonana TR:TREATMENT_SUMMARY cultures grown for 6 days were inoculated with bacterial cells washed in L1 TR:TREATMENT_SUMMARY medium three times (final concentration, 106 bacterial cells mL-1). Co-cultures TR:TREATMENT_SUMMARY were pre-incubated for two days. Test of model prediction experiment – T. TR:TREATMENT_SUMMARY pseudonana was inoculated into L1 as described above. Triplicate samples were TR:TREATMENT_SUMMARY inoculated with three heterotrophic bacteria (R. pomeroyi DSS-3, TR:TREATMENT_SUMMARY Stenotrophomonas sp. SKA-14, and Polaribacter dokdonensis MED-152). Another set TR:TREATMENT_SUMMARY of triplicate samples was kept axenic (diatom only). The cultures were TR:TREATMENT_SUMMARY maintained under 16 h of light period (160 µmol photons m-2 s-1) and 8 h of TR:TREATMENT_SUMMARY dark period cycles. TR:TREATMENT_PROTOCOL_FILENAME uchimiya_20201018_3_Treatment_protocol_UGA_phytoplankton_Oct2020.docx TR:TREATMENT_PROTOCOL_COMMENTS Treatment protocol is in 3_Treatment protocol_UGA_phytoplankton_Oct2020.docx TR:TREATMENT Test of model prediction experiment: with/without bacteria TR:PLANT_LIGHT_PERIOD Light period: 16 hr, dark period: 8 hr TR:PLANT_TEMP 18oC #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Phytoplankton cells were removed from filters using a sonicator SLPe (Branson) SP:SAMPLEPREP_SUMMARY in ultra-pure water (Millipore), concentrated by a lyophilizer (Labconco), and SP:SAMPLEPREP_SUMMARY kept -80oC until further processing. The samples were mixed with 600 µL of 30 SP:SAMPLEPREP_SUMMARY mmol L-1 sodium phosphate buffer (18 mmol L-1 NaHPO4, 12 mmol L-1, pH 7.4) and SP:SAMPLEPREP_SUMMARY an internal standard of 2,2-dimethyl-2-silapentane-5-sulfonate-d6 (DSS, 1 mmol SP:SAMPLEPREP_SUMMARY L-1), vortexed at 4oC for 5 minutes, and centrifuged at 20,800 rcf using an SP:SAMPLEPREP_SUMMARY ultracentrifuge (Eppendorf) at 4oC for 10 minutes, and supernatants were SP:SAMPLEPREP_SUMMARY transferred to 5-mm NMR tubes (Bruker). For the test of model prediction SP:SAMPLEPREP_SUMMARY experiment, 300 µL of supernatants were further diluted with 250 µL of the SP:SAMPLEPREP_SUMMARY buffer and transferred to NMR tubes. All the sample preparation steps were done SP:SAMPLEPREP_SUMMARY on ice or in a cold room (4oC). SP:SAMPLEPREP_PROTOCOL_FILENAME uchimiya_20201018_4_Sample_preparation_protocol_UGA_phytoplankton_Oct2020.docx SP:SAMPLEPREP_PROTOCOL_COMMENTS Sample preparation protocol is in 4_Sample preparation SP:SAMPLEPREP_PROTOCOL_COMMENTS protocol_UGA_phytoplankton_Oct2020.docx SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Water SP:EXTRACT_ENRICHMENT Lyophilization SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Sodium phosphate buffer #CHROMATOGRAPHY #ANALYSIS AN:LABORATORY_NAME Complex Carbohydrate Research Center AN:ANALYSIS_TYPE NMR AN:ANALYSIS_PROTOCOL_FILE 5_Analysis protocol_UGA_phytoplankton_Oct2020.docx AN:ACQUISITION_PARAMETERS_FILE 6_Acquisition and processing parameters_UGA_phytoplankton_Oct2020.xlsx AN:SOFTWARE_VERSION TopSpin 3.5.7, 4.0.1, and 4.0.4 AN:OPERATOR_NAME Mario Uchimiya AN:PROCESSING_PARAMETERS_FILE 6_Acquisition and processing parameters_UGA_phytoplankton_Oct2020.xlsx AN:DATA_FORMAT Bruker #NMR NM:INSTRUMENT_NAME Bruker Avance lll NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 2D-1H-13C NM:NMR_COMMENTS Analysis protocol is in 5_Analysis protocol_UGA_phytoplankton_Oct2020.docx; NM:NMR_COMMENTS detailed aquisition and processing parameters are in 6_Acquisition and NM:NMR_COMMENTS processing parameters_UGA_phytoplankton_Oct2020.xlsx NM:STANDARD_CONCENTRATION 1 mmol L-1 NM:SPECTROMETER_FREQUENCY 600 and 800 MHz NM:NMR_PROBE 800 MHz 5 mm TCI, 800 MHz 1.7 mm TCI and 600 MHz 5 mm TXI NM:NMR_SOLVENT Sodium phosphate buffer in D2O NM:NMR_TUBE_SIZE 5 mm NM:SHIMMING_METHOD TopShim NM:PULSE_SEQUENCE hsqcetgpprsisp2.2, hsqcdietgpsisp.2, and hmbcetgpl2nd NM:CHEMICAL_SHIFT_REF_STD DSS NM:BINNED_DATA_PROTOCOL_FILE Folder 'rNMR' #END