#METABOLOMICS WORKBENCH Tom_Metz_20140709_9836221_mwtab.txt DATATRACK_ID:137 STUDY_ID:ST000085 ANALYSIS_ID:AN000137 PROJECT_ID:PR000075 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Systems Biology for EnteroPathogens PR:PROJECT_TYPE MS analysis PR:PROJECT_SUMMARY sysbep.org PR:INSTITUTE Pacific Northwest National Laboratory PR:DEPARTMENT Biological Separation and Mass Spectrometry PR:LAST_NAME Joshua PR:FIRST_NAME Adkins PR:ADDRESS - PR:EMAIL Joshua.Adkins@pnnl.gov PR:PHONE - #STUDY ST:STUDY_TITLE Salmonella Modulates Metabolism during Growth under Conditions that Induce ST:STUDY_TITLE of Virulence Genes ST:STUDY_TYPE growth conditions, timecourse ST:STUDY_SUMMARY Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative ST:STUDY_SUMMARY that uses complex mechanisms to invade and proliferate within mammalian host ST:STUDY_SUMMARY To investigate possible contributions of metabolic processes to virulence in S. ST:STUDY_SUMMARY grown under conditions known to induce expression of virulence genes, we used a ST:STUDY_SUMMARY systems biology approach coupled with genome scale modeling. First, we ST:STUDY_SUMMARY distinct metabolite profiles associated with bacteria grown in either rich or ST:STUDY_SUMMARY media and report the most comprehensive coverage of the S. Typhimurium ST:STUDY_SUMMARY to date. Second, we applied an omics-informed genome scale modeling analysis of ST:STUDY_SUMMARY functional consequences of adaptive alterations in S. Typhimurium metabolism ST:STUDY_SUMMARY growth under our conditions. Modeling efforts highlighted a decreased cellular ST:STUDY_SUMMARY to both produce and utilize intracellular amino acids during stationary phase ST:STUDY_SUMMARY in virulence conditions, despite significant abundance increases for these ST:STUDY_SUMMARY as observed by our metabolomics measurements. Furthermore, analyses of omics ST:STUDY_SUMMARY in the context of the metabolic model indicated rewiring of the metabolic ST:STUDY_SUMMARY to support pathways associated with virulence. For example, cellular ST:STUDY_SUMMARY of polyamines were perturbed, as well as the predicted capacity for secretion ST:STUDY_SUMMARY uptake. ST:INSTITUTE Pacific Northwest National Laboratory ST:DEPARTMENT Biological Separation and Mass Spectrometry ST:LAST_NAME Metz ST:FIRST_NAME Thomas ST:ADDRESS - ST:EMAIL thomas.metz@pnnl.gov ST:PHONE - ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 18 #SUBJECT SU:SUBJECT_TYPE Cells SU:SUBJECT_SPECIES Salmonella typhimurium SU:TAXONOMY_ID 90371 SU:GENOTYPE_STRAIN CDC 6516-60 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC 14028 SU:SPECIES_GROUP Microorganism #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - SBEP_STM_LB_1 Growth Conditions:Luria-Bertani (LB) medium | Time Harvested:>2.5hour SUBJECT_SAMPLE_FACTORS - SBEP_STM_LB_2 Growth Conditions:Luria-Bertani (LB) medium | Time Harvested:>2.5hour SUBJECT_SAMPLE_FACTORS - SBEP_STM_LB_3 Growth Conditions:Luria-Bertani (LB) medium | Time Harvested:>2.5hour SUBJECT_SAMPLE_FACTORS - SBEP_STM_LPM4_1 Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:4hour SUBJECT_SAMPLE_FACTORS - SBEP_STM_LPM4_2 Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:4hour SUBJECT_SAMPLE_FACTORS - SBEP_STM_LPM4_3 Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:4hour SUBJECT_SAMPLE_FACTORS - SBEP_STM_LPM20_1 Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:20hour SUBJECT_SAMPLE_FACTORS - SBEP_STM_LPM20_2 Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:20hour SUBJECT_SAMPLE_FACTORS - SBEP_STM_LPM20_3 Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:20hour #COLLECTION CO:COLLECTION_SUMMARY Cells were harvested via centrifugation (4,000×g) and washed twice with CO:COLLECTION_SUMMARY PBS (Mediatech) CO:SAMPLE_TYPE Cell CO:COLLECTION_METHOD Centrifugation (4,000 x g) CO:COLLECTION_FREQUENCY Once CO:COLLECTION_TIME variable, see Treatments CO:VOLUMEORAMOUNT_COLLECTED All CO:STORAGE_CONDITIONS -80° C #TREATMENT TR:TREATMENT_SUMMARY Luria-Bertani (LB) medium, hartvested after >2.5 hours | Low pH, low magnesium, TR:TREATMENT_SUMMARY iron (LPM) medium, harvested after 20 h | Low pH, low magnesium, low iron (LPM) TR:TREATMENT_SUMMARY harvested after 4 h | Low pH, low magnesium, low iron (LPM) medium, harvested TR:TREATMENT_SUMMARY 20 h | Low pH, low magnesium, low iron (LPM) medium, harvested after 4 h TR:TREATMENT_PROTOCOL_COMMENTS S. Typhimurium wild type cells (ATCC 14028) were used throughout this study and TR:TREATMENT_PROTOCOL_COMMENTS taken from a single colony on an agar plate, subsequently inoculated into 7 mL TR:TREATMENT_PROTOCOL_COMMENTS Luria-Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast extract, and 1% TR:TREATMENT_PROTOCOL_COMMENTS chloride at pH 7), and incubated overnight at 37°C. The overnight culture was TR:TREATMENT_PROTOCOL_COMMENTS and the supernatant discarded. The cell pellets were suspended in LB media and TR:TREATMENT_PROTOCOL_COMMENTS again, and the supernatant was discarded. For LB cultures, the cell pellets TR:TREATMENT_PROTOCOL_COMMENTS subsequently suspended in 2 mL of LB media and used to inoculate 700 mL of LB TR:TREATMENT_PROTOCOL_COMMENTS a 2.8 L flask. After 160 min of growth, cells were harvested via centrifugation TR:TREATMENT_PROTOCOL_COMMENTS and washed twice with Dulbeccos PBS (Mediatech) once cultures reached an TR:TREATMENT_PROTOCOL_COMMENTS of +1.0. / S. Typhimurium wild type cells (ATCC 14028) were used throughout TR:TREATMENT_PROTOCOL_COMMENTS study and were taken from a single colony on an agar plate, subsequently TR:TREATMENT_PROTOCOL_COMMENTS into 7 mL of Luria-Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast TR:TREATMENT_PROTOCOL_COMMENTS and 1% sodium chloride at pH 7), and incubated overnight at 37°C. The TR:TREATMENT_PROTOCOL_COMMENTS culture was centrifuged and the supernatant discarded. The cell pellets were TR:TREATMENT_PROTOCOL_COMMENTS in LB media and centrifuged again, and the supernatant was discarded. To TR:TREATMENT_PROTOCOL_COMMENTS the Salmonella virulence program, cells were transferred to a low pH, low TR:TREATMENT_PROTOCOL_COMMENTS and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 TR:TREATMENT_PROTOCOL_COMMENTS KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM TR:TREATMENT_PROTOCOL_COMMENTS and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8. Briefly, cell pellets TR:TREATMENT_PROTOCOL_COMMENTS the 7 mL LB media were suspended in LPM media and centrifuged. The supernatant TR:TREATMENT_PROTOCOL_COMMENTS discarded and cell pellets were suspended in 2 mL of LPM media and used to TR:TREATMENT_PROTOCOL_COMMENTS 700 mL of LPM in a 2.8 L flask. Cells were harvested after 20 h, then cells TR:TREATMENT_PROTOCOL_COMMENTS harvested via centrifugation (4,000×g) and washed twice with Dulbeccos PBS TR:TREATMENT_PROTOCOL_COMMENTS Three biological replicates were performed for each of the conditions described TR:TREATMENT_PROTOCOL_COMMENTS S. Typhimurium wild type cells (ATCC 14028) were used throughout this study and TR:TREATMENT_PROTOCOL_COMMENTS taken from a single colony on an agar plate, subsequently inoculated into 7 mL TR:TREATMENT_PROTOCOL_COMMENTS Luria-Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast extract, and 1% TR:TREATMENT_PROTOCOL_COMMENTS chloride at pH 7), and incubated overnight at 37°C. The overnight culture was TR:TREATMENT_PROTOCOL_COMMENTS and the supernatant discarded. The cell pellets were suspended in LB media and TR:TREATMENT_PROTOCOL_COMMENTS again, and the supernatant was discarded. To stimulate the Salmonella virulence TR:TREATMENT_PROTOCOL_COMMENTS cells were transferred to a low pH, low magnesium, and low iron (LPM) medium TR:TREATMENT_PROTOCOL_COMMENTS of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, TR:TREATMENT_PROTOCOL_COMMENTS glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM TR:TREATMENT_PROTOCOL_COMMENTS acid at pH 5.8. Briefly, cell pellets from the 7 mL LB media were suspended in TR:TREATMENT_PROTOCOL_COMMENTS media and centrifuged. The supernatant was discarded and cell pellets were TR:TREATMENT_PROTOCOL_COMMENTS in 2 mL of LPM media and used to inoculate 700 mL of LPM in a 2.8 L flask. TR:TREATMENT_PROTOCOL_COMMENTS were harvested after 4 h, then cells were harvested via centrifugation TR:TREATMENT_PROTOCOL_COMMENTS and washed twice with Dulbeccos PBS (Mediatech). Three biological replicates TR:TREATMENT_PROTOCOL_COMMENTS performed for each of the conditions described above. || S. Typhimurium wild TR:TREATMENT_PROTOCOL_COMMENTS cells (ATCC 14028) were used throughout this study and were taken from a single TR:TREATMENT_PROTOCOL_COMMENTS on an agar plate, subsequently inoculated into 7 mL of Luria-Bertani (LB) media TR:TREATMENT_PROTOCOL_COMMENTS of 1% tryptone, 0.5% yeast extract, and 1% sodium chloride at pH 7), and TR:TREATMENT_PROTOCOL_COMMENTS overnight at 37°C. The overnight culture was centrifuged and the supernatant TR:TREATMENT_PROTOCOL_COMMENTS The cell pellets were suspended in LB media and centrifuged again, and the TR:TREATMENT_PROTOCOL_COMMENTS was discarded. To stimulate the Salmonella virulence program, cells were TR:TREATMENT_PROTOCOL_COMMENTS to a low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, TR:TREATMENT_PROTOCOL_COMMENTS µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, TR:TREATMENT_PROTOCOL_COMMENTS thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH TR:TREATMENT_PROTOCOL_COMMENTS Briefly, cell pellets from the 7 mL LB media were suspended in LPM media and TR:TREATMENT_PROTOCOL_COMMENTS The supernatant was discarded and cell pellets were suspended in 2 mL of LPM TR:TREATMENT_PROTOCOL_COMMENTS and used to inoculate 700 mL of LPM in a 2.8 L flask. Cells were harvested TR:TREATMENT_PROTOCOL_COMMENTS 20 h, then cells were harvested via centrifugation (4,000×g) and washed twice TR:TREATMENT_PROTOCOL_COMMENTS Dulbeccos PBS (Mediatech). Three biological replicates were performed for TR:TREATMENT_PROTOCOL_COMMENTS of the conditions described above. || S. Typhimurium wild type cells (ATCC TR:TREATMENT_PROTOCOL_COMMENTS were used throughout this study and were taken from a single colony on an agar TR:TREATMENT_PROTOCOL_COMMENTS subsequently inoculated into 7 mL of Luria-Bertani (LB) media (comprised of 1% TR:TREATMENT_PROTOCOL_COMMENTS 0.5% yeast extract, and 1% sodium chloride at pH 7), and incubated overnight at TR:TREATMENT_PROTOCOL_COMMENTS The overnight culture was centrifuged and the supernatant discarded. The cell TR:TREATMENT_PROTOCOL_COMMENTS were suspended in LB media and centrifuged again, and the supernatant was TR:TREATMENT_PROTOCOL_COMMENTS To stimulate the Salmonella virulence program, cells were transferred to a low TR:TREATMENT_PROTOCOL_COMMENTS low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM TR:TREATMENT_PROTOCOL_COMMENTS citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% TR:TREATMENT_PROTOCOL_COMMENTS 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8. TR:TREATMENT_PROTOCOL_COMMENTS cell pellets from the 7 mL LB media were suspended in LPM media and TR:TREATMENT_PROTOCOL_COMMENTS The supernatant was discarded and cell pellets were suspended in 2 mL of LPM TR:TREATMENT_PROTOCOL_COMMENTS and used to inoculate 700 mL of LPM in a 2.8 L flask. Cells were harvested TR:TREATMENT_PROTOCOL_COMMENTS 4 h, then cells were harvested via centrifugation (4,000×g) and washed twice TR:TREATMENT_PROTOCOL_COMMENTS Dulbeccos PBS (Mediatech). Three biological replicates were performed for TR:TREATMENT_PROTOCOL_COMMENTS of the conditions described above. TR:TREATMENT Abiotic TR:TREATMENT_ROUTE Media TR:CELL_GROWTH_CONTAINER 700 mL of LB in a 2.8 L flask TR:CELL_MEDIA Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 1% sodium TR:CELL_MEDIA at pH 7) / low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM TR:CELL_MEDIA 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol TR:CELL_MEDIA 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid TR:CELL_MEDIA pH 5.8/ low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM TR:CELL_MEDIA 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol TR:CELL_MEDIA 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid TR:CELL_MEDIA pH 5.8 || low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM TR:CELL_MEDIA 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol TR:CELL_MEDIA 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid TR:CELL_MEDIA pH 5.8 || low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM TR:CELL_MEDIA 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol TR:CELL_MEDIA 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid TR:CELL_MEDIA pH 5.8 TR:CELL_ENVIR_COND 37° C TR:CELL_HARVESTING centrifugation (4,000×g) and washed twice with Dulbeccos PBS (Mediatech) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cell pellets resuspended in ammonium bicarbonate, lysed via bead beating, SP:SAMPLEPREP_SUMMARY with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v) SP:SAMPLEPREP_PROTOCOL_COMMENTS Cell pellets were stored at -80°C prior to thawing and were subsequently SP:SAMPLEPREP_PROTOCOL_COMMENTS in an appropriate volume of 100 mM ammonium bicarbonate according to their wet SP:SAMPLEPREP_PROTOCOL_COMMENTS to compensate for any differences in cell numbers. The cells were lysed by SP:SAMPLEPREP_PROTOCOL_COMMENTS and the lysates were transferred into new tubes. Subsequently, 100 µL aliquots SP:SAMPLEPREP_PROTOCOL_COMMENTS cell lysates were extracted with four volumes of chilled (-20°C) SP:SAMPLEPREP_PROTOCOL_COMMENTS (2:1, v/v), and the aqueous phases after centrifugation were transferred to SP:SAMPLEPREP_PROTOCOL_COMMENTS vials and dried in vacuo (SpeedVac; Thermo Scientific, Waltham, MA). All SP:SAMPLEPREP_PROTOCOL_COMMENTS were kept at -80°C prior to chemical derivatization for GC-MS analysis. Dried SP:SAMPLEPREP_PROTOCOL_COMMENTS extracts were briefly dried again to remove any residual water prior to SP:SAMPLEPREP_PROTOCOL_COMMENTS derivatization. To protect carbonyl groups and reduce the number of tautomeric SP:SAMPLEPREP_PROTOCOL_COMMENTS 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, SP:SAMPLEPREP_PROTOCOL_COMMENTS by incubation at 37°C with generous shaking for 90 min. To derivatize hydroxyl SP:SAMPLEPREP_PROTOCOL_COMMENTS amine groups to trimethylsilyated (TMS) forms, 80 µL of SP:SAMPLEPREP_PROTOCOL_COMMENTS (MSTFA) with 1% trimethylchlorosilane (TMCS) were added to each vial, followed SP:SAMPLEPREP_PROTOCOL_COMMENTS incubation at 37°C with shaking for 30 min. The samples were allowed to cool SP:SAMPLEPREP_PROTOCOL_COMMENTS room temperature and were then analyzed by GC-MS in random order. For technical SP:SAMPLEPREP_PROTOCOL_COMMENTS each of the derivatized samples was split into two different vials and analyzed SP:PROCESSING_METHOD Lysed via bead-beating SP:EXTRACTION_METHOD four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v), and the SP:EXTRACTION_METHOD phases after centrifugation were transferred to glass vials and dried in vacuo SP:EXTRACTION_METHOD Thermo Scientific, Waltham, MA) SP:EXTRACT_ENRICHMENT dried in vacuo SP:EXTRACT_STORAGE dried in vacuo, stored at -80° C SP:SAMPLE_RESUSPENSION 20 µL of methoxyamine in pyridine (30 mg/mL) SP:SAMPLE_DERIVATIZATION 20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of SP:SAMPLE_DERIVATIZATION (MSTFA) with 1% trimethylchlorosilane (TMCS) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Agilent 7890A gas chromatograph with a HP-5MS gas chromatography column using CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent HP5-MS (30m × 0.25mm, 0.25 um) CH:CHROMATOGRAPHY_COMMENTS Chromatography was carried out on an Agilent 7890A gas chromatograph using the CH:CHROMATOGRAPHY_COMMENTS software (Chemstation) and a HP-5MS gas chromatography column (Agilent CH:CHROMATOGRAPHY_COMMENTS Santa Clara, CA; 30 m x 0.25 mm x 0.25 m film thickness). The sample injection CH:CHROMATOGRAPHY_COMMENTS was splitless, and 1 L of each sample was injected. The injection port CH:CHROMATOGRAPHY_COMMENTS was held at 250 C throughout the analysis. The GC oven was held at 60 C for 1 CH:CHROMATOGRAPHY_COMMENTS after injection, and the temperature was then increased to 325 C by 10 C/min, CH:CHROMATOGRAPHY_COMMENTS by a 5 min hold at 325 C. The helium gas flow rates for each Experiment were CH:CHROMATOGRAPHY_COMMENTS by the Agilent Retention Time Locking function based on analysis of deuterated CH:CHROMATOGRAPHY_COMMENTS acid and were in the range of 0.450.5 mL/min. CH:FLOW_RATE 0.450.5 mL/min CH:INJECTION_TEMPERATURE 250 C CH:SAMPLE_INJECTION 1 L, splitless CH:ANALYTICAL_TIME 37.5 min CH:OVEN_TEMPERATURE 60 C for 1 min, then increased to 325 C by 10 C/min, followed by a 5 min hold CH:OVEN_TEMPERATURE 325 C CH:SAMPLE_SYRINGE_SIZE 10 L #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Biological Separations & Mass Spectrometry group, Pacific Northwest National AN:LABORATORY_NAME (www.omics.pnl.gov) AN:ACQUISITION_DATE 2013 AN:SOFTWARE_VERSION Metabolite Detector vs. 2.0.6 beta AN:DATA_FORMAT Raw .D.Zip; Processed .CDF #MS MS:INSTRUMENT_NAME Agilent 5975C MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS An Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent MS:MS_COMMENTS Inc.; Santa Clara, CA, USA) was used, and the samples were blocked and analyzed MS:MS_COMMENTS random order for each experiment. Data were collected over the mass range MS:MS_COMMENTS m/z. A mixture of FAMEs (C8-C28) was analyzed once per day together with the MS:MS_COMMENTS for retention index alignment purposes during subsequent data analysis. MS:SCAN_RANGE_MOVERZ 50-550 m/z MS:MS_COMMENTS After converting raw data to netCDF format, the data were processed by the MS:MS_COMMENTS software for peak deconvolution and chromatographic alignment. Retention MS:MS_COMMENTS (RI) were calculated based on the analysis of a mixture of fatty acid methyl MS:MS_COMMENTS (C8 - C30) (Agilent Technologies) as external retention time standards, then MS:MS_COMMENTS retention index information was subsequently applied to all experimental MS:MS_COMMENTS for retention time alignment. MetaboliteDetector parameters for peak detection MS:MS_COMMENTS deconvolution are as follows: Peak threshold, 7; minimum peak height, 7; MS:MS_COMMENTS width, 8. Deconvoluted features were identified by matching to the Agilent MS:MS_COMMENTS Metabolomics Retention Time Locked Library, which contains mass spectral and MS:MS_COMMENTS index information for approximately 700 metabolites. Each initial match to the MS:MS_COMMENTS was manually inspected to confirm a confident identification. MS:MS_COMMENTS software was used for database matching and batch identification/quantification MS:MS_COMMENTS are as follows: required score, 0.6; ?RI, 25; minimum S/N, 20; maximum peak MS:MS_COMMENTS index, 100. Ions 73 and 143 were excluded from use as metabolite quantification MS:MS_COMMENTS since these are due to fragmentation of the trimethylsilyl groups. Otherwise, MS:MS_COMMENTS unique fragment ions were assigned to each metabolite for quantification and MS:MS_COMMENTS for each individual GC-MS analysis when processing the data in batch mode. The MS:MS_COMMENTS areas of the three quantification ions were exported from MetaboliteDetector MS:MS_COMMENTS used in further statistical anayses. All identifications were manually MS:MS_COMMENTS by inspection of retention index and spectrum matches. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples SBEP_STM_LPM20_1 SBEP_STM_LPM20_2 SBEP_STM_LPM20_3 SBEP_STM_LPM4_1 SBEP_STM_LPM4_2 SBEP_STM_LPM4_3 SBEP_STM_LB_1 SBEP_STM_LB_2 SBEP_STM_LB_3 Factors Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:20hour Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:20hour Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:20hour Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:4hour Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:4hour Growth Conditions:Low pH, low magnesium, low iron (LPM) medium | Time Harvested:4hour Growth Conditions:Luria-Bertani (LB) medium | Time Harvested:>2.5hour Growth Conditions:Luria-Bertani (LB) medium | Time Harvested:>2.5hour Growth Conditions:Luria-Bertani (LB) medium | Time Harvested:>2.5hour 1_3-diaminopropane -0.8151 -0.8151 -0.8151 -0.4872 -0.4165 -0.4080 0.5053 1.7966 1.4550 2_6-diaminopimelic acid 0.8067 0.4197 1.7093 -1.2563 -0.5660 -0.4104 -0.7029 4-guanidinobutyric acid 1.2770 0.2133 -1.1369 1.5736 -0.5359 -0.9536 -0.6065 0.1691 4-hydroxyphenyllactic acid 0.8083 0.7868 -0.3016 1.4041 0.5216 -1.2329 -0.9296 -1.0568 6-deoxy-D-glucose 0.4991 0.0379 1.9583 -0.2122 -0.5355 -1.3416 -0.4060 adenine 0.6688 0.4967 0.7652 -1.4048 -1.1574 -1.1710 1.0892 1.0248 -0.3115 adenosine 1.4963 0.9465 -0.9398 -1.0292 -0.4634 0.7424 -0.7527 adenosine-5-monophosphate 0.2399 1.4880 0.7501 -0.8923 -1.4422 -1.0615 0.5478 0.9629 -0.5928 alpha-D-glucose -0.0007 -0.1520 0.0029 0.2198 0.5450 2.1132 -0.9478 -0.8804 -0.9001 beta- alanine -0.4220 -0.8393 -0.3217 -0.5981 -0.1294 -0.9882 -0.9960 1.4658 1.1790 citramalic acid -0.0417 -0.2003 0.0523 0.0055 -0.7103 2.1107 -1.1688 0.2579 -0.3054 citrulline -0.4958 1.3357 0.5173 -0.6108 -0.7972 -1.2357 -0.1762 0.7401 0.7226 cytosine 0.9691 1.0507 1.6213 -0.8693 -0.8567 -0.4617 -0.5969 -0.8566 deoxycytidylic acid 0.8879 1.0751 1.4480 -1.3043 -1.0120 -0.4173 0.0001 -0.6776 D-glucose-6-phosphate -0.7211 -0.7547 -1.0250 -0.5527 -0.3395 -0.3500 0.8807 1.7747 1.0877 DIHYDROXYACETONE PHOSPHATE -0.6143 -0.6143 -0.6143 -0.6143 -0.6143 -0.6143 1.4046 1.7228 0.5587 diphosphoric acid 1.8681 1.0318 0.2420 -0.6127 -0.6435 -0.4433 0.0641 -0.7345 -0.7719 DL-glyceraldehyde -0.7740 -0.9591 -1.0004 0.8874 -0.7770 1.0558 1.6384 -0.5825 0.5115 D-malic acid -0.3458 -0.6455 0.1117 0.0374 2.1149 1.1193 -1.1320 -0.8612 -0.3989 D-ribose -0.2910 0.6346 1.2631 1.6755 -0.1449 -0.9291 -0.5495 -0.6294 -1.0293 fructose -0.3667 -0.6945 -0.7057 -0.0040 -0.4423 -0.4996 0.3330 2.3797 glyceric acid 0.3605 0.7544 2.2651 -0.1851 -0.1902 -0.2864 -0.8953 -0.8915 -0.9314 glycerol -0.1640 -0.3460 -0.4620 -0.2992 -0.5362 2.0234 0.8836 -1.0997 glycine 0.9445 1.1288 1.1504 -0.1852 0.4052 0.2497 -1.3162 -0.9401 -1.4372 glycolic acid -0.8101 -0.4881 -0.7364 -0.6834 -0.5774 -0.1842 0.4638 1.5486 1.4673 hypoxanthine -0.9087 -0.7461 -0.5466 0.2793 1.7421 0.9634 -0.6335 0.8478 -0.9976 L-alanine 0.9796 0.5948 1.2726 -0.3153 0.8540 0.2706 -1.3086 -1.2656 -1.0819 L-aspartic acid 0.7107 0.6658 1.6841 -0.5988 0.6352 0.2410 -1.1470 -1.0896 -1.1012 L-cysteine 0.7638 1.3258 1.8077 -0.2568 -0.7002 -0.4086 -0.8266 -0.8746 -0.8305 L-glutamic acid 1.2034 1.0302 1.6607 -0.8570 0.0755 -0.7248 -0.8974 -0.6370 -0.8536 L-histidine 0.6885 0.8051 2.0955 -0.6347 -0.1311 -0.5085 -0.7819 -0.7746 -0.7584 L-homoserine 0.2378 0.3465 -0.1260 2.2977 -0.5577 -0.8751 -0.5352 -0.7880 l-isoleucine 0.2510 0.7887 0.7437 -0.4565 1.4969 0.0328 -1.2667 -0.9688 -0.6211 L-Lactic acid -0.1320 0.2367 -0.2837 -0.4914 0.5593 1.7982 -0.4009 -0.5737 -0.7126 L-leucine 0.7908 1.1784 1.4674 -0.7868 0.6712 -0.2961 -0.8441 -1.0826 -1.0982 L-lysine 1.1212 1.6210 1.2729 -0.4493 -0.4188 -0.5861 -0.8674 -0.8333 -0.8603 L-methionine 0.5830 0.9997 1.3201 -0.5080 1.1508 -0.2203 -1.0274 -1.2483 -1.0495 L-ornithine 0.9830 1.3981 0.8242 -0.6115 0.9624 -0.4876 -0.8756 -1.1706 -1.0224 L-proline 0.9086 0.6674 1.0047 0.4259 -0.5837 -1.3130 -1.1100 L-serine 0.6130 1.6498 1.4532 -0.4366 -0.0323 -0.5736 -0.9471 -0.8466 -0.8797 L-threonine -0.0481 -0.6791 1.1847 0.9911 1.2843 -0.9711 -0.8332 -0.9286 L-tyrosine 0.9475 1.3365 1.3630 -0.6117 0.2949 -0.2072 -0.9898 -1.0343 -1.0989 L-valine -0.1128 0.1048 0.2414 0.3239 1.4364 1.4817 -1.1155 -1.1673 -1.1926 malonic acid -0.6978 -0.9579 -0.7910 -0.3364 -0.6907 -0.3715 1.6000 1.4209 0.8243 myristic acid 1.1328 1.2988 -0.5073 -1.4735 -0.9041 -0.1974 0.3279 0.3229 N-Acetyl-L-glutamic acid 0.8164 1.5489 0.7062 -0.6362 -0.5123 0.4575 -1.2107 -1.1697 nicotinic acid 0.1099 -0.0165 0.5248 -1.7909 -0.4043 -1.1032 0.0889 1.2792 1.3122 oleic acid -0.5170 -0.2345 -0.7863 -0.4852 -0.3395 -0.6643 -0.0082 0.7885 2.2464 palmitic acid -0.4124 0.0497 -1.0050 -0.1229 0.6370 0.2536 -0.6288 0.8203 0.4085 palmitoleic acid -1.0391 -0.6443 -0.7880 -0.5112 -0.2053 -0.5984 1.7959 0.5415 1.4489 pantothenic acid 1.9153 -0.1484 -0.0171 0.4213 0.7315 0.3992 -1.2031 -0.9039 -1.1946 phosphoenolpyruvic acid 0.6431 0.9786 1.2569 -0.8937 -0.8017 -0.2931 0.9799 -0.4885 -1.3816 phosphoric acid -1.5508 -0.5121 0.5114 -0.1643 0.0325 0.8010 0.1827 -0.1463 0.8460 porphine -0.4398 1.2126 0.1447 0.1524 0.2009 -0.2884 -0.8540 0.3294 -0.4579 putrescine 1.0459 0.9125 0.9067 -0.0508 0.7092 0.3708 -1.3714 -1.3215 -1.2015 pyruvic acid -0.5869 -0.9072 -1.3398 0.3460 -0.6857 -0.3809 1.2095 1.1046 1.2405 sn-Glycerol 3-phosphate -0.5928 -0.5959 -0.5963 -0.4772 -0.4924 -0.5181 2.0998 1.4717 -0.2987 spermidine -0.5771 -0.6150 -0.6218 -0.8123 -0.6606 -0.7666 1.6841 1.3093 1.0602 stearic acid -0.1611 0.7695 -0.2985 0.1904 0.2134 1.0492 -1.4793 0.5935 -0.8770 succinic acid -0.1839 -0.1990 0.3816 0.7150 1.1439 0.9379 -2.0470 -0.7242 -0.0243 thymine 0.6511 0.5845 0.8739 -0.4676 1.4206 0.3351 -0.7980 -0.8775 -1.7221 tyramine 0.0495 0.7014 1.4236 -1.4490 -0.6377 -1.4001 0.1968 0.2547 0.8608 uracil -0.2800 -0.3439 -0.0868 0.4620 1.6491 1.1339 -0.9007 0.0408 -1.6743 urea 0.4792 0.9191 0.6882 1.1291 -1.2274 -0.8506 -1.1376 urocanic acid 0.4785 1.0263 1.2326 -0.1479 0.6501 0.3742 -1.2046 -1.2046 -1.2046 xanthine 1.0845 1.1829 1.4620 -0.3455 -0.2362 -0.8877 -0.2969 -1.0097 -0.9534 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name moverz_quant ri ri_type pubchem_id inchi_key kegg_id other_id other_id_type 1,3-diaminopropane 428 CHEBI:15725 PNNL_ID 2,6-diaminopimelic acid 865 CHEBI:23673 PNNL_ID 4-guanidinobutyric acid 500 CHEBI:15728 PNNL_ID 4-hydroxyphenyllactic acid 9378 CHEBI:17385 PNNL_ID 6-deoxy-D-glucose 93579 CHEBI:28140 PNNL_ID adenine 62648 CHEBI:16708 PNNL_ID adenosine CHEBI:16335 PNNL_ID adenosine-5-monophosphate CHEBI:16027 PNNL_ID alpha-D-glucose CHEBI:17925 PNNL_ID beta- alanine CHEBI:16958 PNNL_ID citramalic acid 1081 CHEBI:15584 PNNL_ID citrulline 9750 CHEBI:16349 PNNL_ID cytosine CHEBI:16040 PNNL_ID deoxycytidylic acid 13945 CHEBI:15918 PNNL_ID D-glucose-6-phosphate 439427 CID44134741 PNNL_ID DIHYDROXYACETONE PHOSPHATE 668 CHEBI:57642 PNNL_ID diphosphoric acid 21961011 CHEBI:29888 PNNL_ID DL-glyceraldehyde 751 CHEBI:15693 PNNL_ID D-malic acid 92824 CHEBI:30796 PNNL_ID D-ribose 441481 CHEBI:47014 PNNL_ID fructose 5984 CHEBI:48095 PNNL_ID glyceric acid 752 CHEBI:32398 PNNL_ID glycerol 11126194 CHEBI:17522 PNNL_ID glycine 750 CHEBI:15428 PNNL_ID glycolic acid 757 CHEBI:17497 PNNL_ID hypoxanthine CHEBI:17368 PNNL_ID L-alanine 5950 CHEBI:16977 PNNL_ID L-aspartic acid 5960 CHEBI:17053 PNNL_ID L-cysteine 5862 CHEBI:32736 PNNL_ID L-glutamic acid 33032 CHEBI:16015 PNNL_ID L-histidine 6274 CHEBI:15971 PNNL_ID L-homoserine CHEBI:15699 PNNL_ID l-isoleucine 99288 CHEBI:17191 PNNL_ID L-Lactic acid 107689 CHEBI:422 PNNL_ID L-leucine 6106 CHEBI:28225 PNNL_ID L-lysine 5962 CHEBI:18019 PNNL_ID L-methionine 6137 CHEBI:16643 PNNL_ID L-ornithine CHEBI:15729 PNNL_ID L-proline 145742 CHEBI:17203 PNNL_ID L-serine 5951 CHEBI:17115 PNNL_ID L-threonine 6288 CHEBI:16857 PNNL_ID L-tyrosine 6057 CHEBI:32759 PNNL_ID L-valine 6287 CHEBI:57762 PNNL_ID malonic acid 867 CHEBI:30794 PNNL_ID myristic acid CHEBI:28875 PNNL_ID N-Acetyl-L-glutamic acid 185 CHEBI:17533 PNNL_ID nicotinic acid 938 CHEBI:15940 PNNL_ID oleic acid 965 CHEBI:16196 PNNL_ID palmitic acid 5326436 CHEBI:15756 PNNL_ID palmitoleic acid 5282745 CHEBI:59265 PNNL_ID pantothenic acid 11777341 CHEBI:46905 PNNL_ID phosphoenolpyruvic acid 11754241 CHEBI:44897 PNNL_ID phosphoric acid 1004 CHEBI:26078 PNNL_ID porphine 5481287 CHEBI:8337 PNNL_ID putrescine 1045 CHEBI:17148 PNNL_ID pyruvic acid CHEBI:32816 PNNL_ID sn-Glycerol 3-phosphate 439162 CHEBI:15978 PNNL_ID spermidine CHEBI:16610 PNNL_ID stearic acid CHEBI:25629 PNNL_ID succinic acid 8036 CHEBI:15741 PNNL_ID thymine 1135 CHEBI:17821 PNNL_ID tyramine 5610 CHEBI:15760 PNNL_ID uracil 12212752 CHEBI:17568 PNNL_ID urea CHEBI:16199 PNNL_ID urocanic acid CHEBI:30817 PNNL_ID xanthine CHEBI:17712 PNNL_ID METABOLITES_END #END