{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002433","ANALYSIS_ID":"AN003963","VERSION":"1","CREATED_ON":"02-08-2024"},

"PROJECT":{"PROJECT_TITLE":"Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale modeling","PROJECT_SUMMARY":"Anaerobic microbial metabolism drives critical functions within global ecosystems, host-microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance a versatile approach to elaborate cellular metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an amino acid and carbohydrate-fermenting Clostridia. High-Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy of C. difficile, grown with fermentable 13C substrates, informed dynamic flux balance analysis (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic recruitment of oxidative and supporting reductive pathways, with integration of high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to support efficient energy generation, nitrogen handling, and biomass generation. Model predictions informed an approach leveraging the sensitivity of 13C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow from [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. Findings identify metabolic strategies used by C. difficile to support its rapid colonization and expansion in gut ecosystems.","INSTITUTE":"Brigham and Women's Hospital","DEPARTMENT":"Pathology","LABORATORY":"Bry Lab (Massachusetts Host-Microbiome Center, BWH) and Cheng Lab (Massachusetts General Hospital)","LAST_NAME":"Pavao","FIRST_NAME":"Aidan","ADDRESS":"221 Longwood Ave, EBRC-411, Boston, MA, 02115, USA","EMAIL":"apavao2@bwh.harvard.edu","PHONE":"617-525-7184","FUNDING_SOURCE":"NIH R01AI153653, R03AI174158, P30DK056338, S10OD023406, R21CA243255, and R01AG070257; BWH Precision Medicine Institute; MGH A. A. Martinos Center for Biomedical Imaging","PUBLICATIONS":"https://doi.org/10.1038/s41589-023-01275-9","DOI":"http://dx.doi.org/10.21228/M88M5G","CONTRIBUTORS":"Aidan Pavao, Brintha Girinathan, Johann Peltier, Pamela Altamirano Silva, Bruno Dupuy, Isabella H. Muti, Craig Malloy, Leo L. Cheng, Lynn Bry"},

"STUDY":{"STUDY_TITLE":"Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale modeling","STUDY_SUMMARY":"Anaerobic microbial metabolism drives critical functions within global ecosystems, host-microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance a versatile approach to elaborate cellular metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an amino acid and carbohydrate-fermenting Clostridia. High-Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy of C. difficile, grown with fermentable 13C substrates, informed dynamic flux balance analysis (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic recruitment of oxidative and supporting reductive pathways, with integration of high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to support efficient energy generation, nitrogen handling, and biomass generation. Model predictions informed an approach leveraging the sensitivity of 13C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow from [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. Findings identify metabolic strategies used by C. difficile to support its rapid colonization and expansion in gut ecosystems.","INSTITUTE":"Brigham and Women's Hospital","DEPARTMENT":"Pathology","LABORATORY":"Bry Lab, Massachusetts Host-Microbiome Center; Cheng Lab, Massachusetts General Hospital","LAST_NAME":"Pavao","FIRST_NAME":"Aidan","ADDRESS":"221 Longwood Ave, EBRC-411, Boston, MA, 02115, USA","EMAIL":"apavao2@bwh.harvard.edu","PHONE":"617-525-7184","SUBMIT_DATE":"2023-01-05"},

"SUBJECT":{"SUBJECT_TYPE":"Bacteria","SUBJECT_SPECIES":"Clostridioides difficile","TAXONOMY_ID":"NCBI:txid1496","GENOTYPE_STRAIN":"ATCC 43255 delPaLoc"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Data10_13CGlc_endpt1",
"Factors":{"Condition":"13C-Glucose"},
"Additional sample data":{"RAW_FILE_NAME":"Data10_13CGlc_endpt1","Type":"Culture supernatant","Analysis":"Alanine 14N:15N"}
},
{
"Subject ID":"-",
"Sample ID":"Data11_13CGlc_endpt2",
"Factors":{"Condition":"13C-Glucose"},
"Additional sample data":{"RAW_FILE_NAME":"Data11_13CGlc_endpt2","Type":"Culture supernatant","Analysis":"Alanine 14N:15N"}
},
{
"Subject ID":"-",
"Sample ID":"Data12_13CGlc_endpt3",
"Factors":{"Condition":"13C-Glucose"},
"Additional sample data":{"RAW_FILE_NAME":"Data12_13CGlc_endpt3","Type":"Culture supernatant","Analysis":"Alanine 14N:15N"}
},
{
"Subject ID":"-",
"Sample ID":"Data7_13CGlc1",
"Factors":{"Condition":"13C-Glucose"},
"Additional sample data":{"RAW_FILE_NAME":"Data7_13CGlc1","Type":"Time series","Analysis":"dFBA, Alanine 14N:15N"}
},
{
"Subject ID":"-",
"Sample ID":"Data8_13CGlc2",
"Factors":{"Condition":"13C-Glucose"},
"Additional sample data":{"RAW_FILE_NAME":"Data8_13CGlc2","Type":"Time series","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data9_13CGlc3",
"Factors":{"Condition":"13C-Glucose"},
"Additional sample data":{"RAW_FILE_NAME":"Data9_13CGlc3","Type":"Time series","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data19_13CGlc_standards",
"Factors":{"Condition":"13C-Glucose, 13C-Acetate, 13C-Alanine, 13C-Ethanol, 13C-Butyrate"},
"Additional sample data":{"RAW_FILE_NAME":"Data19_13CGlc_standards","Type":"Chemical solutions","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data13_13CGlc_15NLeu_endpt1",
"Factors":{"Condition":"13C-Glucose, 15N-Leucine"},
"Additional sample data":{"RAW_FILE_NAME":"Data13_13CGlc_15NLeu_endpt1","Type":"Culture supernatant","Analysis":"Alanine 14N:15N"}
},
{
"Subject ID":"-",
"Sample ID":"Data14_13CGlc_15NLeu_endpt2",
"Factors":{"Condition":"13C-Glucose, 15N-Leucine"},
"Additional sample data":{"RAW_FILE_NAME":"Data14_13CGlc_15NLeu_endpt2","Type":"Culture supernatant","Analysis":"Alanine 14N:15N"}
},
{
"Subject ID":"-",
"Sample ID":"Data15_13CGlc_15NLeu_endpt3",
"Factors":{"Condition":"13C-Glucose, 15N-Leucine"},
"Additional sample data":{"RAW_FILE_NAME":"Data15_13CGlc_15NLeu_endpt3","Type":"Culture supernatant","Analysis":"Alanine 14N:15N"}
},
{
"Subject ID":"-",
"Sample ID":"Data16_13CGlc_15NLeu_stack",
"Factors":{"Condition":"13C-Glucose, 15N-Leucine"},
"Additional sample data":{"RAW_FILE_NAME":"Data16_13CGlc_15NLeu_stack","Type":"Time series","Analysis":"Alanine 14N:15N"}
},
{
"Subject ID":"-",
"Sample ID":"Data4_13CLeu1",
"Factors":{"Condition":"13C-Leucine"},
"Additional sample data":{"RAW_FILE_NAME":"Data4_13CLeu1","Type":"Time series","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data5_13CLeu2",
"Factors":{"Condition":"13C-Leucine"},
"Additional sample data":{"RAW_FILE_NAME":"Data5_13CLeu2","Type":"Time series","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data6_13CLeu3",
"Factors":{"Condition":"13C-Leucine"},
"Additional sample data":{"RAW_FILE_NAME":"Data6_13CLeu3","Type":"Time series","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data1_13CPro1",
"Factors":{"Condition":"13C-Proline"},
"Additional sample data":{"RAW_FILE_NAME":"Data1_13CPro1","Type":"Time series","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data2_13CPro2",
"Factors":{"Condition":"13C-Proline"},
"Additional sample data":{"RAW_FILE_NAME":"Data2_13CPro2","Type":"Time series","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data3_13CPro3",
"Factors":{"Condition":"13C-Proline"},
"Additional sample data":{"RAW_FILE_NAME":"Data3_13CPro3","Type":"Time series","Analysis":"dFBA"}
},
{
"Subject ID":"-",
"Sample ID":"Data20_2D",
"Factors":{"Condition":"13C-Proline, 13C-Leucine, 13C-Glucose"},
"Additional sample data":{"RAW_FILE_NAME":"Data20_2D","Type":"2D time series snapshots","Analysis":"Metabolite identification"}
},
{
"Subject ID":"-",
"Sample ID":"Data18_13CPro-Se",
"Factors":{"Condition":"13C-Proline, no Selenium source"},
"Additional sample data":{"RAW_FILE_NAME":"Data18_13CPro-Se","Type":"Time series","Analysis":"Selenium perturbation"}
},
{
"Subject ID":"-",
"Sample ID":"Data17_13CPro+Se",
"Factors":{"Condition":"13C-Proline, Sodium Selenite"},
"Additional sample data":{"RAW_FILE_NAME":"Data17_13CPro+Se","Type":"Time series","Analysis":"Selenium perturbation"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For live cell time series, labeled MMM with 10% D2O was inoculated with 100,000 vegetative Clostridioides difficile ATCC43255 delPaLoc cells in a Kel-F HRMAS rotor insert under anaerobic atmosphere. The sample was spun at 3600 Hz at 37°C in a 4mm zirconia rotor and successive spectra were acquired over 36+ hours. For static culture supernatants, labeled MMM was inoculated with 100,000 vegetative Clostridioides difficile ATCC43255 delPaLoc cells in an anaerobic chamber and incubated at 37°C for 48 hours. Cultures were centrifuged and supernatants collected and lyophilized, then resuspended in D2O prior to NMR spectra acquisition.","SAMPLE_TYPE":"Bacterial cells","VOLUMEORAMOUNT_COLLECTED":"30 µL","COLLECTION_VIALS":"Kel-F inserts for 4mm MAS rotor","COLLECTION_TUBE_TEMP":"37°C"},

"TREATMENT":{"TREATMENT_SUMMARY":"For live cell time series, cells were grown in MMM with 10% D2O and one or more labeled substrates (L-[U-13C]Proline, L-[U-13C]Leucine, [U-13C]Glucose, or both [U-13C]Glucose and L-[15N]Leucine). For culture supernatants, cells were grown in MMM with [U-13C]Glucose, with or without L-[15N]Leucine. For the selenium perturbation time series, cells were grown in MMM with 10% L-[U-13C]Proline, with or without 100µM sodium selenite.","TREATMENT_COMPOUND":"L-[U-13C]Proline, L-[U-13C]Leucine, [U-13C]Glucose, L-[15N]Leucine, sodium selenite","CELL_MEDIA":"C. difficile Modified Minimal Medium (MMM) with 100µM sodium selenite and 10% D2O","CELL_ENVIR_COND":"anaerobic, 37°C"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"For live cell NMR time series, samples were added to the HRMAS rotor neat. For culture supernatants, lyophilized samples were resuspended in D2O added to the NMR rotor neat."},

"CHROMATOGRAPHY":[],

"ANALYSIS":{"LABORATORY_NAME":"Cheng Lab","ANALYSIS_TYPE":"NMR","ACQUISITION_PARAMETERS_FILE":"acqus","SOFTWARE_VERSION":"TopSpin 3.6.2","OPERATOR_NAME":"Leo L Cheng","PROCESSING_PARAMETERS_FILE":"pdata/1/procs","DATA_FORMAT":"Bruker"},

"NM":{"INSTRUMENT_NAME":"Bruker Avance III HD","INSTRUMENT_TYPE":"FT-NMR","NMR_EXPERIMENT_TYPE":"Other","NMR_COMMENTS":"Experiments collected 1D 1H, 1D 13C, 2D 1H, 2D 13C, and 2D 1H-13C data.","SPECTROMETER_FREQUENCY":"600 MHz","NMR_SOLVENT":"C. difficile Modified Minimal Medium (MMM) with 100µM sodium selenite and 10% D2O","TEMPERATURE":"37"},

"NMR_METABOLITE_DATA":{
"Units":"proportion of Alanine",

"Data":[{"Metabolite":"[2_3-13C_ 14N]Alanine","Data10_13CGlc_endpt1":"0.4700","Data11_13CGlc_endpt2":"0.5300","Data12_13CGlc_endpt3":"0.5300","Data13_13CGlc_15NLeu_endpt1":"0.1700","Data14_13CGlc_15NLeu_endpt2":"0.2200","Data15_13CGlc_15NLeu_endpt3":"0.2400"},{"Metabolite":"[2_3-13C_ 15N]Alanine","Data10_13CGlc_endpt1":"","Data11_13CGlc_endpt2":"","Data12_13CGlc_endpt3":"","Data13_13CGlc_15NLeu_endpt1":"0.2800","Data14_13CGlc_15NLeu_endpt2":"0.2600","Data15_13CGlc_15NLeu_endpt3":"0.2600"},{"Metabolite":"[U-13C_ 14N]Alanine","Data10_13CGlc_endpt1":"0.5300","Data11_13CGlc_endpt2":"0.4700","Data12_13CGlc_endpt3":"0.4700","Data13_13CGlc_15NLeu_endpt1":"0.1900","Data14_13CGlc_15NLeu_endpt2":"0.2400","Data15_13CGlc_15NLeu_endpt3":"0.2300"},{"Metabolite":"[U-13C_ 15N]Alanine","Data10_13CGlc_endpt1":"","Data11_13CGlc_endpt2":"","Data12_13CGlc_endpt3":"","Data13_13CGlc_15NLeu_endpt1":"0.3600","Data14_13CGlc_15NLeu_endpt2":"0.2700","Data15_13CGlc_15NLeu_endpt3":"0.2700"}],

"Metabolites":[{"Metabolite":"[2,3-13C, 14N]Alanine","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""},{"Metabolite":"[2,3-13C, 15N]Alanine","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""},{"Metabolite":"[U-13C, 14N]Alanine","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""},{"Metabolite":"[U-13C, 15N]Alanine","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""}]
}

}