#METABOLOMICS WORKBENCH uchimiya_20210831_081703 DATATRACK_ID:2818 STUDY_ID:ST001944 ANALYSIS_ID:AN003166 PROJECT_ID:PR001231 VERSION 1 CREATED_ON September 6, 2021, 2:57 pm #PROJECT PR:PROJECT_TITLE Growth-stage related diatom-bacteria interactions PR:PROJECT_SUMMARY Phytoplankton-derived metabolites fuel a large fraction of heterotrophic PR:PROJECT_SUMMARY bacterial production in the global ocean, yet methodological challenges have PR:PROJECT_SUMMARY limited our knowledge of organic molecules transferred between these two PR:PROJECT_SUMMARY microbial groups. In an experimental bloom study in which the diatom PR:PROJECT_SUMMARY Thalassiosira pseudonana was co-cultured with three heterotrophic marine PR:PROJECT_SUMMARY bacteria, we concurrently measured diatom endometabolites (i.e., potential PR:PROJECT_SUMMARY exometabolite supply) by nuclear magnetic resonance (NMR) spectroscopy and PR:PROJECT_SUMMARY bacterial gene expression (i.e., potential exometabolite uptake) by PR:PROJECT_SUMMARY metatranscriptomic sequencing. PR:INSTITUTE University of Georgia PR:DEPARTMENT Department of Marine Sciences; Complex Carbohydrate Research Center PR:LABORATORY Moran Lab, Edison Lab PR:LAST_NAME Mario PR:FIRST_NAME Uchimiya PR:ADDRESS 315 Riverbend Rd, Athens, GA 30602 PR:EMAIL mario.uchimiya@uga.edu PR:PHONE (706) 542-8387 PR:FUNDING_SOURCE National Science Foundation (OCE-1948104); Swedish Research Council (2018-06571) PR:CONTRIBUTORS Malin Olofsson #STUDY ST:STUDY_TITLE Growth-stage related diatom-bacteria interactions ST:STUDY_SUMMARY Phytoplankton-derived metabolites fuel a large fraction of heterotrophic ST:STUDY_SUMMARY bacterial production in the global ocean, yet methodological challenges have ST:STUDY_SUMMARY limited our knowledge of organic molecules transferred between these two ST:STUDY_SUMMARY microbial groups. In an experimental bloom study in which the diatom ST:STUDY_SUMMARY Thalassiosira pseudonana was co-cultured with three heterotrophic marine ST:STUDY_SUMMARY bacteria, we concurrently measured diatom endometabolites (i.e., potential ST:STUDY_SUMMARY exometabolite supply) by nuclear magnetic resonance (NMR) spectroscopy and ST:STUDY_SUMMARY bacterial gene expression (i.e., potential exometabolite uptake) by ST:STUDY_SUMMARY metatranscriptomic sequencing. ST:INSTITUTE University of Georgia ST:DEPARTMENT Department of Marine Sciences; Complex Carbohydrate Research Center ST:LABORATORY Moran Lab, Edison Lab ST:LAST_NAME Mario ST:FIRST_NAME Uchimiya ST:ADDRESS 315 Riverbend Rd, Athens, GA 30602 ST:EMAIL mario.uchimiya@uga.edu ST:PHONE (706) 542-8387 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Thalassiosira pseudonana SU:TAXONOMY_ID 296543 SU:GENOTYPE_STRAIN CCMP1335 #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 Endometabolome 61 Factor-1:Early Growth phase | Factor-2:With bacteria Sample_descritpion=Phytoplankton-bacteria co-culture day 3; RAW_FILE_NAME=61; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=1; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 5 2019; Alternative_identifications_in_the_study=Flask 6 SUBJECT_SAMPLE_FACTORS Endometabolome 15 Factor-1:Early Growth phase | Factor-2:With bacteria Sample_descritpion=Phytoplankton-bacteria co-culture day 3; RAW_FILE_NAME=15; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=2; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 5 2019; Alternative_identifications_in_the_study=Flask 12 SUBJECT_SAMPLE_FACTORS Endometabolome 17 Factor-1:Early Growth phase | Factor-2:With bacteria Sample_descritpion=Phytoplankton-bacteria co-culture day 3; RAW_FILE_NAME=17; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=3; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 5 2019; Alternative_identifications_in_the_study=Flask 13 SUBJECT_SAMPLE_FACTORS Endometabolome 25 Factor-1:Late Growth phase | Factor-2:With bacteria Sample_descritpion=Phytoplankton-bacteria co-culture day 15; RAW_FILE_NAME=25; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=1; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 17 2019; Alternative_identifications_in_the_study=Flask 5 SUBJECT_SAMPLE_FACTORS Endometabolome 27 Factor-1:Late Growth phase | Factor-2:With bacteria Sample_descritpion=Phytoplankton-bacteria co-culture day 15; RAW_FILE_NAME=27; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=2; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 17 2019; Alternative_identifications_in_the_study=Flask 7 SUBJECT_SAMPLE_FACTORS Endometabolome 29 Factor-1:Late Growth phase | Factor-2:With bacteria Sample_descritpion=Phytoplankton-bacteria co-culture day 15; RAW_FILE_NAME=29; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=3; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 17 2019; Alternative_identifications_in_the_study=Flask 8 SUBJECT_SAMPLE_FACTORS Endometabolome 31 Factor-1:Late Growth phase | Factor-2:Without bacteria Sample_descritpion=Phytoplankton Axenic day 15; RAW_FILE_NAME=31; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=1; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 17 2019; Alternative_identifications_in_the_study=Flask 18 A SUBJECT_SAMPLE_FACTORS Endometabolome 33 Factor-1:Late Growth phase | Factor-2:Without bacteria Sample_descritpion=Phytoplankton Axenic day 15; RAW_FILE_NAME=33; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=2; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 17 2019; Alternative_identifications_in_the_study=Flask 20 A SUBJECT_SAMPLE_FACTORS Endometabolome 35 Factor-1:Late Growth phase | Factor-2:Without bacteria Sample_descritpion=Phytoplankton Axenic day 15; RAW_FILE_NAME=35; Organism=Phytoplankton, marine diatom, Thalassiosira pseudonana CCMP1335; Experiment_name=Diatom growth-stages; Biological_replicate=3; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=October 17 2019; Alternative_identifications_in_the_study=Flask 21 A SUBJECT_SAMPLE_FACTORS Blank 43 Factor-1:NA | Factor-2:NA Sample_descritpion=Medium blank; RAW_FILE_NAME=43; Organism=NA; Experiment_name=NA; Biological_replicate=NA; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=NA; Alternative_identifications_in_the_study=– SUBJECT_SAMPLE_FACTORS Blank 51 Factor-1:NA | Factor-2:NA Sample_descritpion=Medium blank; RAW_FILE_NAME=51; Organism=NA; Experiment_name=NA; Biological_replicate=NA; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=NA; Alternative_identifications_in_the_study=– SUBJECT_SAMPLE_FACTORS Blank 57 Factor-1:NA | Factor-2:NA Sample_descritpion=Medium blank; RAW_FILE_NAME=57; Organism=NA; Experiment_name=NA; Biological_replicate=NA; NMR_probe=600 MHz 5mm TCI; NMR_experiment=HSQC; Acquisition_setting_identification_number=Acquistion-1; Sample_collection_date=NA; Alternative_identifications_in_the_study=– #COLLECTION CO:COLLECTION_SUMMARY During this synthetic bloom experiment, axenic cultures of the diatom CO:COLLECTION_SUMMARY Thalssiosira pseudonana CCMP1335 (National Center for Marine Algae) were CO:COLLECTION_SUMMARY inoculated with equal cell numbers of the heterotrophic bacteria Ruegeria CO:COLLECTION_SUMMARY pomeroyi DSS-3 (Rhodobacterales), Stenotrophomonas sp. SKA-14 (Xanthomonadales), CO:COLLECTION_SUMMARY and Polaribacter dokdonensis MED-152 (Flavobacteriales) and co-cultured for 20 CO:COLLECTION_SUMMARY d. Co-cultured diatoms were harvested at day 3 and day 15, as well as axenic CO:COLLECTION_SUMMARY cultures (diatom only) day 15. Subsamples of 700-1000 ml were filtered onto 2.0 CO:COLLECTION_SUMMARY µm pore-size Isopore (Millipore, Burlington, MA) filters. Filters were stored CO:COLLECTION_SUMMARY in 50 ml falcon tubes at -80°C until processing. CO:COLLECTION_PROTOCOL_FILENAME 2_Collection protocol_UGA_phytoplankton_ Aug2021.docx CO:COLLECTION_PROTOCOL_COMMENTS Details are in 2_Collection protocol_UGA_phytoplankton_ Aug2021.docx. CO:SAMPLE_TYPE Algae CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The diatom was grown in organic-carbon free L1 medium (Guillard and Hargraves TR:TREATMENT_SUMMARY 1993) prepared in acid washed glass containers at a salinity of 35 (Harrison et TR:TREATMENT_SUMMARY al. 1980) for one week prior to the start of the experiment. Cultures were grown TR:TREATMENT_SUMMARY with a 16:8 h light:dark cycle under 160 µmol photons m-2 s-1 at 18°C and TR:TREATMENT_SUMMARY checked for bacterial contamination by plating on rich medium (½ YTSS). On Day TR:TREATMENT_SUMMARY 0 of the experiment, diatoms were transferred into 1.9 L culture flasks TR:TREATMENT_SUMMARY containing 1000 ml of medium made with 13C-bicarbonate (final concentration of ~ TR:TREATMENT_SUMMARY 2x103 cells ml-1). One flask was kept as an L1 medium control without organisms. TR:TREATMENT_SUMMARY The three strains of heterotrophic bacteria were grown overnight in rich medium TR:TREATMENT_SUMMARY made with artificial seawater, either ½ YTSS medium (R. pomeroyi and TR:TREATMENT_SUMMARY Stenotrophomonas) or 1/10 YTSS medium (P. dokdonensis). Cells were harvested in TR:TREATMENT_SUMMARY exponential growth phase and washed five times in the same artificial sea water TR:TREATMENT_SUMMARY used for preparing the L1 medium. The bacteria were inoculated in equal TR:TREATMENT_SUMMARY proportions of OD600 into 15 flasks containing diatoms, with a final combined TR:TREATMENT_SUMMARY concentration of ca. 1x105 cells ml-1. One set of three flasks remained axenic. TR:TREATMENT_SUMMARY Three co-culture flasks were sacrificed at 4 pm days 3 and 15, and the axenic TR:TREATMENT_SUMMARY flasks day 15. TR:TREATMENT_PROTOCOL_FILENAME 3_Treatment protocol_UGA_phytoplankton_ Aug2021.docx TR:TREATMENT_PROTOCOL_COMMENTS Details are in 3_Treatment protocol_UGA_phytoplankton_ Aug2021.docx. TR:TREATMENT Incubation of a diatom strain with and without bacterial strains #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, 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). All the sample preparation steps were SP:SAMPLEPREP_SUMMARY done on ice or in a cold room (4oC). SP:SAMPLEPREP_PROTOCOL_FILENAME 4_Sample preparation protocol_UGA_phytoplankton_ Aug2021.docx SP:SAMPLEPREP_PROTOCOL_COMMENTS Details are in 4_Sample preparation protocol_UGA_phytoplankton_ Aug2021.docx. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Water SP:EXTRACT_ENRICHMENT Lyophilization SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Sodium phosphate buffer #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Complex Carbohydrate Research Center AN:ANALYSIS_PROTOCOL_FILE 5_Analysis protocol_UGA_phytoplankton_ Aug2021.docx AN:ACQUISITION_PARAMETERS_FILE 6_Acquisition and processing parameters_UGA_phytoplankton_ Aug2021.xlsx AN:PROCESSING_PARAMETERS_FILE 6_Acquisition and processing parameters_UGA_phytoplankton_ Aug2021.xlsx AN:DATA_FORMAT Bruker #NMR NM:INSTRUMENT_NAME AVANCE Ⅲ NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 2D-1H-13C NM:NMR_COMMENTS Details are in 5_Analysis protocol_UGA_phytoplankton_ Aug2021.docx. All the NM:NMR_COMMENTS experiment and processing parameters are in 6_Acquisition and processing NM:NMR_COMMENTS parameters_UGA_phytoplankton_ Aug2021.xlsx. NM:STANDARD_CONCENTRATION 1 mmol L-1 NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE 5 mm TXI NM:NMR_SOLVENT Sodium phosphate buffer NM:NMR_TUBE_SIZE 5 mm NM:SHIMMING_METHOD topshim NM:PULSE_SEQUENCE hsqcetgpprsisp2.2; hsqcdietgpsisp.2 NM:WATER_SUPPRESSION Yes NM:CHEMICAL_SHIFT_REF_CPD 2,2-dimethyl-2-silapentane-5-sulfonate-d6 NM:TEMPERATURE 25 NM:NMR_RESULTS_FILE ST001944_AN003166_Results.txt UNITS:Intensity #END