#METABOLOMICS WORKBENCH lichenjie1996_20251114_045931 DATATRACK_ID:6695 STUDY_ID:ST004351 ANALYSIS_ID:AN007263 PROJECT_ID:PR002760 VERSION 1 CREATED_ON November 15, 2025, 12:23 pm #PROJECT PR:PROJECT_TITLE Interactions with bacteria shape diatom adaptation to carbon concentration PR:PROJECT_TITLE changes PR:PROJECT_SUMMARY Diatoms are key contributors to global primary production, and have developed PR:PROJECT_SUMMARY intricate partnerships with bacteria through long-term co-evolution. Here, we PR:PROJECT_SUMMARY uncover a syntrophic relationship between the model obligate photoautotroph PR:PROJECT_SUMMARY Phaeodactylum tricornutum and Loktanella vestfoldensis, which enables the diatom PR:PROJECT_SUMMARY to indirectly utilize glucose. Reanalysis of Tara Oceans metagenomic data shows PR:PROJECT_SUMMARY frequent co-occurrence of Loktanella with diatoms including Chaetoceros and PR:PROJECT_SUMMARY Thalassiosira, indicating the ecological relevance of this partnership. PR:PROJECT_SUMMARY Co-culture with L. vestfoldensis supports robust growth of Chaetoceros muelleri PR:PROJECT_SUMMARY and Thalassiosira pseudonana in the presence of glucose as the sole carbon PR:PROJECT_SUMMARY source. Transcriptomic and metabolomic analyses reveal that P. tricornutum PR:PROJECT_SUMMARY maintains a photoautotrophic metabolism in co-culture, as indicated by the PR:PROJECT_SUMMARY up-regulation of genes involved in inorganic carbon concentration and PR:PROJECT_SUMMARY photosynthesis, while the co-cultured bacterium likely supplies CO2 and PR:PROJECT_SUMMARY growth-stimulating metabolites such as indole-3-acetic acid. Our findings PR:PROJECT_SUMMARY demonstrate that bacterial-algal interactions may shape diatom adaptation to PR:PROJECT_SUMMARY carbon changes and contribute to marine carbon cycling. PR:INSTITUTE Chinese Academy of Sciences PR:DEPARTMENT Institute of Hydrobiology PR:LAST_NAME Li PR:FIRST_NAME Chenjie PR:ADDRESS No. 7 Donghu South Road, Wuhan City, Hubei Province, 430072, China PR:EMAIL 1159958986@qq.com PR:PHONE 86-027-68780078 #STUDY ST:STUDY_TITLE Interactions with bacteria shape diatom adaptation to carbon concentration ST:STUDY_TITLE changes ST:STUDY_SUMMARY Diatoms are key contributors to global primary production, and have developed ST:STUDY_SUMMARY intricate partnerships with bacteria through long-term co-evolution. Here, we ST:STUDY_SUMMARY uncover a syntrophic relationship between the model obligate photoautotroph ST:STUDY_SUMMARY Phaeodactylum tricornutum and Loktanella vestfoldensis, which enables the diatom ST:STUDY_SUMMARY to indirectly utilize glucose. Reanalysis of Tara Oceans metagenomic data shows ST:STUDY_SUMMARY frequent co-occurrence of Loktanella with diatoms including Chaetoceros and ST:STUDY_SUMMARY Thalassiosira, indicating the ecological relevance of this partnership. ST:STUDY_SUMMARY Co-culture with L. vestfoldensis supports robust growth of Chaetoceros muelleri ST:STUDY_SUMMARY and Thalassiosira pseudonana in the presence of glucose as the sole carbon ST:STUDY_SUMMARY source. Transcriptomic and metabolomic analyses reveal that P. tricornutum ST:STUDY_SUMMARY maintains a photoautotrophic metabolism in co-culture, as indicated by the ST:STUDY_SUMMARY up-regulation of genes involved in inorganic carbon concentration and ST:STUDY_SUMMARY photosynthesis, while the co-cultured bacterium likely supplies CO2 and ST:STUDY_SUMMARY growth-stimulating metabolites such as indole-3-acetic acid. Our findings ST:STUDY_SUMMARY demonstrate that bacterial-algal interactions may shape diatom adaptation to ST:STUDY_SUMMARY carbon changes and contribute to marine carbon cycling. ST:INSTITUTE Chinese Academy of Sciences ST:DEPARTMENT Institute of Hydrobiology ST:LAST_NAME Li ST:FIRST_NAME Chenjie ST:ADDRESS No. 7 Donghu South Road, Wuhan City, Hubei Province, 430072, China ST:EMAIL 1159958986@qq.com ST:PHONE 86-027-68780078 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Phaeodactylum tricornutum, Loktanella vestfoldensis SU:TAXONOMY_ID 2850, 245188 #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 - Co-culture_1 group:Co-culture | Sample source:Supernatant RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-Co-culture_1.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-Co-culture_1.raw SUBJECT_SAMPLE_FACTORS - Co-culture_2 group:Co-culture | Sample source:Supernatant RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-Co-culture_2.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-Co-culture_2.raw SUBJECT_SAMPLE_FACTORS - Co-culture_3 group:Co-culture | Sample source:Supernatant RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-Co-culture_3.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-Co-culture_3.raw SUBJECT_SAMPLE_FACTORS - Monoculture_1 group:Monoculture | Sample source:Supernatant RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-Monoculture_1.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-Monoculture_1.raw SUBJECT_SAMPLE_FACTORS - Monoculture_2 group:Monoculture | Sample source:Supernatant RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-Monoculture_2.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-Monoculture_2.raw SUBJECT_SAMPLE_FACTORS - Monoculture_3 group:Monoculture | Sample source:Supernatant RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-Monoculture_3.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-Monoculture_3.raw SUBJECT_SAMPLE_FACTORS - QC1 group:QC | Sample source:Supernatant mixture RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-QC1.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-QC1.raw SUBJECT_SAMPLE_FACTORS - QC2 group:QC | Sample source:Supernatant mixture RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-QC2.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-QC2.raw SUBJECT_SAMPLE_FACTORS - QC3 group:QC | Sample source:Supernatant mixture RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-QC3.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-QC3.raw SUBJECT_SAMPLE_FACTORS - QC4 group:QC | Sample source:Supernatant mixture RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-QC4.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-QC4.raw SUBJECT_SAMPLE_FACTORS - Blank group:Blank | Sample source:Methanol RAW_FILE_NAME(Positive_Raw_File_Name)=Positive-Blank.raw; RAW_FILE_NAME(Negative_Raw_File_Name)=Negative-Blank.raw #COLLECTION CO:COLLECTION_SUMMARY Co-cultured samples were obtained from the supernatant after centrifugation of CO:COLLECTION_SUMMARY Phaeodactylum tricornutum and Loktanella vestfoldensis co-culture medium. CO:COLLECTION_SUMMARY Monocultured samples were obtained from the supernatant after centrifugation of CO:COLLECTION_SUMMARY Loktanella vestfoldensis monoculture medium. QC samples were obtained from a CO:COLLECTION_SUMMARY mixture of co-cultured and monocultured supernatants, and were used to test the CO:COLLECTION_SUMMARY stability of the data. All cell cultures were centrifuged at 2000 × g for 10 CO:COLLECTION_SUMMARY min, and 30 mL supernatant was collected and stored at -80 °C. CO:COLLECTION_PROTOCOL_FILENAME LiChenjie_Protocol.pdf CO:SAMPLE_TYPE Media #TREATMENT TR:TREATMENT_SUMMARY One mL (OD600 ≈ 0.4) of PtCr epiphytic bacterium was inoculated into 50 mL TR:TREATMENT_SUMMARY plug-seal cap culture flasks containing 30 mL of f/2-enriched artificial TR:TREATMENT_SUMMARY seawater (without NaHCO3) with 3 g/L glucose as the sole carbon source, and then TR:TREATMENT_SUMMARY Pt1 (6 × 106 cells) (co-culture) or bicarbonate-free artificial seawater TR:TREATMENT_SUMMARY (designated as “bacterial monoculture”) of an equal volume was added to the TR:TREATMENT_SUMMARY bacterial cultures. Subsequently, the cultures were maintained at 22 °C with TR:TREATMENT_SUMMARY shaking (120 rpm) under continuous illumination of 100 μmol photons m-2 s-1 for TR:TREATMENT_SUMMARY 6 days. TR:TREATMENT_PROTOCOL_FILENAME LiChenjie_Protocol.pdf #SAMPLEPREP SP:SAMPLEPREP_SUMMARY One mL of samples centrifuged at 4 ℃, 13000 rpm for 5 minutes, collect the SP:SAMPLEPREP_SUMMARY supernatant and filter it through a 0.22 µm aqueous membrane. Take 80 µL of SP:SAMPLEPREP_SUMMARY filtrate and add 20 µL of 300 µg/mL internal standard for sample analysis. A SP:SAMPLEPREP_SUMMARY mixture of 200 µL per sample was divided into 4 replicates, and the first three SP:SAMPLEPREP_SUMMARY pooled samples were used to monitor the precision of the instrument, and the SP:SAMPLEPREP_SUMMARY last one was used to monitor the stability of the instrument. SP:SAMPLEPREP_PROTOCOL_FILENAME LiChenjie_Protocol.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The injection volume for each sample was 2 μL, and methanol was used as a blank CH:CHROMATOGRAPHY_SUMMARY sample to deduct baseline features from the mobile phase. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% methanol; 0.1% formic acid CH:FLOW_GRADIENT 0-1 min, 10% B; 1-13 min, linear increase to 98% B; 13-18 min, 98% B; 18-18.5 CH:FLOW_GRADIENT min, linear decrease to 10% B; 18.5-20 min, 10% B CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 300 #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE LiChenjie_Protocol.pdf #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass spectrometry parameters included: capillary temperature, 300 °C; sheath MS:MS_COMMENTS gas flow, 40 arbitrary units; nebulizer temperature, 350 °C. Samples were MS:MS_COMMENTS maintained at 4 °C in the autosampler during analysis. Raw LC-MS data were MS:MS_COMMENTS converted to Analysis Base File (ABF) format using the ABF Converter tool. MS:MS_COMMENTS MS-DIAL software (version 4.70) was used to perform blank deduction (peak MS:MS_COMMENTS intensity [Sample/Blank] >= 3), feature peak screening (at least 2 feature peaks MS:MS_COMMENTS appearing in repeated samples), and normalization (LOWESS method) on the data of MS:MS_COMMENTS positive and negative ion modes separately. A comprehensive feature matrix was MS:MS_COMMENTS generated, containing metabolite identifiers, RT, mass-to-charge ratio (m/z), MS:MS_COMMENTS ion patterns, and peak intensities. Metabolite annotation was achieved by MS:MS_COMMENTS matching experimental RT and m/z values against the MassBank database with the MS:MS_COMMENTS following parameters: RT tolerance = ± 0.1 min, MS1 mass tolerance = 0.005 Da, MS:MS_COMMENTS MS2 mass tolerance = 0.0025 Da, and MS2 spectral match score > 0.7. Missing MS:MS_COMMENTS values were imputed using 20% of the minimum value within each experimental MS:MS_COMMENTS group. MS:MS_RESULTS_FILE ST004351_AN007263_Results.txt UNITS:Peak Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END