#METABOLOMICS WORKBENCH wernerajz_20190302_122938_mwtab.txt DATATRACK_ID:1659 STUDY_ID:ST001196 ANALYSIS_ID:AN001992 PROJECT_ID:PR000808 VERSION 1 CREATED_ON June 18, 2019, 9:20 pm #PROJECT PR:PROJECT_TITLE A comprehensive time-course metabolite profiling of the model cyanobacterium PR:PROJECT_TITLE Synechocystis sp. PCC 6803 under diurnal light:dark cycles PR:PROJECT_SUMMARY Cyanobacteria are a model photoautotroph and a chassis for the sustainable PR:PROJECT_SUMMARY production of fuels and chemicals. Yet, knowledge of photoautotrophic metabolism PR:PROJECT_SUMMARY in the natural environment of day/night cycles is lacking yet has implications PR:PROJECT_SUMMARY for improved yield from plants, algae, and cyanobacteria. Here, a thorough PR:PROJECT_SUMMARY approach to characterizing diverse metabolites—including carbohydrates, PR:PROJECT_SUMMARY lipids, amino acids, pigments, co-factors, nucleic acids and PR:PROJECT_SUMMARY polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. PR:PROJECT_SUMMARY 6803) under sinusoidal diurnal light-dark cycles was developed and applied. A PR:PROJECT_SUMMARY custom photobioreactor and novel multi-platform mass spectrometry workflow PR:PROJECT_SUMMARY enabled metabolite profiling every 30-120 minutes across a 24-hour diurnal PR:PROJECT_SUMMARY sinusoidal LD (“sinLD”) cycle peaking at 1,600 ?mol photons m 2 s-1. We PR:PROJECT_SUMMARY report widespread oscillations across the sinLD cycle with 90%, 94%, and 40% of PR:PROJECT_SUMMARY the identified polar/semi-polar, non-polar, and polymeric metabolites displaying PR:PROJECT_SUMMARY statistically significant oscillations, respectively. Microbial growth displayed PR:PROJECT_SUMMARY distinct lag, biomass accumulation, and cell division phases of growth. During PR:PROJECT_SUMMARY the lag phase, amino acids (AA) and nucleic acids (NA) accumulated to high PR:PROJECT_SUMMARY levels per cell followed by decreased levels during the biomass accumulation PR:PROJECT_SUMMARY phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates PR:PROJECT_SUMMARY displayed sharp oscillations per cell at the day-to-night transition. Potential PR:PROJECT_SUMMARY bottlenecks in central carbon metabolism are highlighted. Together, this report PR:PROJECT_SUMMARY provides a comprehensive view of photosynthetic metabolite behavior with high PR:PROJECT_SUMMARY temporal resolution, offering insight into the impact of growth synchronization PR:PROJECT_SUMMARY to light cycles via circadian rhythms. Incorporation into computational modeling PR:PROJECT_SUMMARY and metabolic engineering efforts promises to improve industrially-relevant PR:PROJECT_SUMMARY strain design. PR:INSTITUTE Colorado State University PR:DEPARTMENT Chemical and PR:LAST_NAME Peebles PR:FIRST_NAME Christie PR:ADDRESS 700 Meridian Ave, Fort Collins, CO 80523 USA PR:EMAIL wernerajz@gmail.com PR:PHONE 2699981811 #STUDY ST:STUDY_TITLE Non-targeted GC-MS Analysis of Insoluble Metabolites (part-II) ST:STUDY_SUMMARY Cyanobacteria are a model photoautotroph and a chassis for the sustainable ST:STUDY_SUMMARY production of fuels and chemicals. Yet, knowledge of photoautotrophic metabolism ST:STUDY_SUMMARY in the natural environment of day/night cycles is lacking yet has implications ST:STUDY_SUMMARY for improved yield from plants, algae, and cyanobacteria. Here, a thorough ST:STUDY_SUMMARY approach to characterizing diverse metabolites—including carbohydrates, ST:STUDY_SUMMARY lipids, amino acids, pigments, co-factors, nucleic acids and ST:STUDY_SUMMARY polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. ST:STUDY_SUMMARY 6803) under sinusoidal diurnal light-dark cycles was developed and applied. A ST:STUDY_SUMMARY custom photobioreactor and novel multi-platform mass spectrometry workflow ST:STUDY_SUMMARY enabled metabolite profiling every 30-120 minutes across a 24-hour diurnal ST:STUDY_SUMMARY sinusoidal LD (“sinLD”) cycle peaking at 1,600 ?mol photons m 2 s-1. We ST:STUDY_SUMMARY report widespread oscillations across the sinLD cycle with 90%, 94%, and 40% of ST:STUDY_SUMMARY the identified polar/semi-polar, non-polar, and polymeric metabolites displaying ST:STUDY_SUMMARY statistically significant oscillations, respectively. Microbial growth displayed ST:STUDY_SUMMARY distinct lag, biomass accumulation, and cell division phases of growth. During ST:STUDY_SUMMARY the lag phase, amino acids (AA) and nucleic acids (NA) accumulated to high ST:STUDY_SUMMARY levels per cell followed by decreased levels during the biomass accumulation ST:STUDY_SUMMARY phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates ST:STUDY_SUMMARY displayed sharp oscillations per cell at the day-to-night transition. Potential ST:STUDY_SUMMARY bottlenecks in central carbon metabolism are highlighted. Together, this report ST:STUDY_SUMMARY provides a comprehensive view of photosynthetic metabolite behavior with high ST:STUDY_SUMMARY temporal resolution, offering insight into the impact of growth synchronization ST:STUDY_SUMMARY to light cycles via circadian rhythms. Incorporation into computational modeling ST:STUDY_SUMMARY and metabolic engineering efforts promises to improve industrially-relevant ST:STUDY_SUMMARY strain design. ST:INSTITUTE Colorado State University ST:DEPARTMENT Chemical and Biological Engineering ST:LAST_NAME Peebles ST:FIRST_NAME Christie ST:ADDRESS 700 Meridian Ave, Fort Collins, CO 80523 ST:EMAIL christie.peebles@colostate.edu ST:PHONE 970-491-6779 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Synechocystis sp. PCC 6803 SU:TAXONOMY_ID 1148 SU:GENOTYPE_STRAIN NCBI:txid1148 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1-Synechocystis_6803-cell-5a-1 time:-2 SUBJECT_SAMPLE_FACTORS - 2-Synechocystis_6803-cell-5a-2 time:-2 SUBJECT_SAMPLE_FACTORS - 3-Synechocystis_6803-cell-5a-3 time:-2 SUBJECT_SAMPLE_FACTORS - 4-Synechocystis_6803-cell-6a-1 time:-1 SUBJECT_SAMPLE_FACTORS - 5-Synechocystis_6803-cell-6a-2 time:-1 SUBJECT_SAMPLE_FACTORS - 6-Synechocystis_6803-cell-6a-3 time:-1 SUBJECT_SAMPLE_FACTORS - 7-Synechocystis_6803-cell-630a-1 time:-0.5 SUBJECT_SAMPLE_FACTORS - 8-Synechocystis_6803-cell-630a-2 time:-0.5 SUBJECT_SAMPLE_FACTORS - 9-Synechocystis_6803-cell-630a-3 time:-0.5 SUBJECT_SAMPLE_FACTORS - 10-Synechocystis_6803-cell-7a-1 time:- SUBJECT_SAMPLE_FACTORS - 11-Synechocystis_6803-cell-7a-2 time:- SUBJECT_SAMPLE_FACTORS - 12-Synechocystis_6803-cell-7a-3 time:- SUBJECT_SAMPLE_FACTORS - 13-Synechocystis_6803-cell-730a-1 time:0.5 SUBJECT_SAMPLE_FACTORS - 14-Synechocystis_6803-cell-730a-2 time:0.5 SUBJECT_SAMPLE_FACTORS - 15-Synechocystis_6803-cell-730a-3 time:0.5 SUBJECT_SAMPLE_FACTORS - 16-Synechocystis_6803-cell-8a-1 time:1 SUBJECT_SAMPLE_FACTORS - 17-Synechocystis_6803-cell-8a-2 time:1 SUBJECT_SAMPLE_FACTORS - 18-Synechocystis_6803-cell-8a-3 time:1 SUBJECT_SAMPLE_FACTORS - 19-Synechocystis_6803-cell-9a-1 time:2 SUBJECT_SAMPLE_FACTORS - 20-Synechocystis_6803-cell-9a-2 time:2 SUBJECT_SAMPLE_FACTORS - 21-Synechocystis_6803-cell-9a-3 time:2 SUBJECT_SAMPLE_FACTORS - 22-Synechocystis_6803-cell-11a-1 time:4 SUBJECT_SAMPLE_FACTORS - 23-Synechocystis_6803-cell-11a-2 time:4 SUBJECT_SAMPLE_FACTORS - 24-Synechocystis_6803-cell-11a-3 time:4 SUBJECT_SAMPLE_FACTORS - 25-Synechocystis_6803-cell-1p-1 time:6 SUBJECT_SAMPLE_FACTORS - 26-Synechocystis_6803-cell-1p-2 time:6 SUBJECT_SAMPLE_FACTORS - 27-Synechocystis_6803-cell-1p-3 time:6 SUBJECT_SAMPLE_FACTORS - 28-Synechocystis_6803-cell-3p-1 time:8 SUBJECT_SAMPLE_FACTORS - 29-Synechocystis_6803-cell-3p-2 time:8 SUBJECT_SAMPLE_FACTORS - 30-Synechocystis_6803-cell-3p-3 time:8 SUBJECT_SAMPLE_FACTORS - 31-Synechocystis_6803-cell-5p-1 time:10 SUBJECT_SAMPLE_FACTORS - 32-Synechocystis_6803-cell-5p-2 time:10 SUBJECT_SAMPLE_FACTORS - 33-Synechocystis_6803-cell-5p-3 time:10 SUBJECT_SAMPLE_FACTORS - 34-Synechocystis_6803-cell-6p-1 time:11 SUBJECT_SAMPLE_FACTORS - 35-Synechocystis_6803-cell-6p-2 time:11 SUBJECT_SAMPLE_FACTORS - 36-Synechocystis_6803-cell-6p-3 time:11 SUBJECT_SAMPLE_FACTORS - 37-Synechocystis_6803-cell-630p-1 time:11.5 SUBJECT_SAMPLE_FACTORS - 38-Synechocystis_6803-cell-630p-2 time:11.5 SUBJECT_SAMPLE_FACTORS - 39-Synechocystis_6803-cell-630p-3 time:11.5 SUBJECT_SAMPLE_FACTORS - 40-Synechocystis_6803-cell-7p-1 time:12 SUBJECT_SAMPLE_FACTORS - 41-Synechocystis_6803-cell-7p-2 time:12 SUBJECT_SAMPLE_FACTORS - 42-Synechocystis_6803-cell-7p-3 time:12 SUBJECT_SAMPLE_FACTORS - 43-Synechocystis_6803-cell-730p-1 time:12.5 SUBJECT_SAMPLE_FACTORS - 44-Synechocystis_6803-cell-730p-2 time:12.5 SUBJECT_SAMPLE_FACTORS - 45-Synechocystis_6803-cell-730p-3 time:12.5 SUBJECT_SAMPLE_FACTORS - 46-Synechocystis_6803-cell-8p-1 time:13 SUBJECT_SAMPLE_FACTORS - 47-Synechocystis_6803-cell-8p-2 time:13 SUBJECT_SAMPLE_FACTORS - 48-Synechocystis_6803-cell-8p-3 time:13 SUBJECT_SAMPLE_FACTORS - 49-Synechocystis_6803-cell-9p-1 time:14 SUBJECT_SAMPLE_FACTORS - 50-Synechocystis_6803-cell-9p-2 time:14 SUBJECT_SAMPLE_FACTORS - 51-Synechocystis_6803-cell-9p-3 time:14 SUBJECT_SAMPLE_FACTORS - 52-Synechocystis_6803-cell-11p-1 time:16 SUBJECT_SAMPLE_FACTORS - 53-Synechocystis_6803-cell-11p-2 time:16 SUBJECT_SAMPLE_FACTORS - 54-Synechocystis_6803-cell-11p-3 time:16 SUBJECT_SAMPLE_FACTORS - 55-Synechocystis_6803-cell-1a-1 time:18 SUBJECT_SAMPLE_FACTORS - 56-Synechocystis_6803-cell-1a-2 time:18 SUBJECT_SAMPLE_FACTORS - 57-Synechocystis_6803-cell-1a-3 time:18 SUBJECT_SAMPLE_FACTORS - 58-Synechocystis_6803-cell-3a-1 time:20 SUBJECT_SAMPLE_FACTORS - 59-Synechocystis_6803-cell-3a-2 time:20 SUBJECT_SAMPLE_FACTORS - 60-Synechocystis_6803-cell-3a-3 time:20 SUBJECT_SAMPLE_FACTORS - 61-Synechocystis_6803-cell-5a_day2-1 time:22 SUBJECT_SAMPLE_FACTORS - 62-Synechocystis_6803-cell-5a_day2-2 time:22 SUBJECT_SAMPLE_FACTORS - 63-Synechocystis_6803-cell-5a_day2-3 time:22 SUBJECT_SAMPLE_FACTORS - 64-Synechocystis_6803-cell-6a_day2-1 time:23 SUBJECT_SAMPLE_FACTORS - 65-Synechocystis_6803-cell-6a_day3-2 time:23 SUBJECT_SAMPLE_FACTORS - 66-Synechocystis_6803-cell-6a_day4-3 time:23 SUBJECT_SAMPLE_FACTORS - 67-Synechocystis_6803-cell-630a_day2-1 time:23.5 SUBJECT_SAMPLE_FACTORS - 68-Synechocystis_6803-cell-630a_day3-2 time:23.5 SUBJECT_SAMPLE_FACTORS - 69-Synechocystis_6803-cell-630a_day4-3 time:23.5 SUBJECT_SAMPLE_FACTORS - 70-Synechocystis_6803-cell-7a_day2-1 time:24 SUBJECT_SAMPLE_FACTORS - 71-Synechocystis_6803-cell-7a_day2-2 time:24 SUBJECT_SAMPLE_FACTORS - 72-Synechocystis_6803-cell-7a_day2-3 time:24 SUBJECT_SAMPLE_FACTORS - QC-1 time:26 SUBJECT_SAMPLE_FACTORS - QC-2 time:26 SUBJECT_SAMPLE_FACTORS - QC-3 time:26 SUBJECT_SAMPLE_FACTORS - QC-4 time:26 SUBJECT_SAMPLE_FACTORS - QC-5 time:26 SUBJECT_SAMPLE_FACTORS - QC-6 time:26 SUBJECT_SAMPLE_FACTORS - QC-7 time:26 SUBJECT_SAMPLE_FACTORS - QC-8 time:26 SUBJECT_SAMPLE_FACTORS - QC-9 time:26 SUBJECT_SAMPLE_FACTORS - QC-10 time:26 SUBJECT_SAMPLE_FACTORS - QC-11 time:26 SUBJECT_SAMPLE_FACTORS - QC-12 time:26 #COLLECTION CO:COLLECTION_SUMMARY For each metabolomics time-point, a 10 mL culture were rapidly sampled via CO:COLLECTION_SUMMARY sterile on-reactor syringes into a pre-weighed centrifuge tube, quenched in CO:COLLECTION_SUMMARY -4°C 1X PBS, spun at 3,000g for 5 min., decanted, frozen in liquid nitrogen, CO:COLLECTION_SUMMARY and lyophilized at -50°C. The workflow from sampling to centrifugation took < 2 CO:COLLECTION_SUMMARY minutes; lyophilized samples were stored at -80°C for < 1 month prior to CO:COLLECTION_SUMMARY extraction. A biphasic extraction from lyophilized cell pellets was performed CO:COLLECTION_SUMMARY via a 2:1:1.6 MTBE:MeOH:H2O biphasic extraction, modified from the protocol CO:COLLECTION_SUMMARY developed by Salem et al. (Salem et al., 2016) resulting in a top layer of MTBE CO:COLLECTION_SUMMARY with non-polar soluble metabolites, a lower layer of MeOH:H2O with polar and CO:COLLECTION_SUMMARY semi-polar soluble metabolites, and an insoluble pellet. Each liquid layer was CO:COLLECTION_SUMMARY transferred to a fresh glass vial and dried under nitrogen gas overnight. The CO:COLLECTION_SUMMARY MTBE layer was resuspended in 1:1 toluene:MeOH and analyzed via Q-TOF-MS with a CO:COLLECTION_SUMMARY UPLC Phenyl Hexyl column (“RP-MS”). The MeOH:H2O layer was resuspended in CO:COLLECTION_SUMMARY 1:1 H2O:MeOH, split evenly and subjected to either i) derivatization in CO:COLLECTION_SUMMARY methoxyamine HCl and MSTFA followed by GC-MS analysis, or ii) targeted SRM CO:COLLECTION_SUMMARY analysis on a tandem quadrupole-MS equipped with a HILIC column. The insoluble CO:COLLECTION_SUMMARY pellet was hydrolyzed with a hydrochloric acid (HCl) based on previously CO:COLLECTION_SUMMARY published protocols (Fountoulakis and Lahm, 1998) (Huang, Kaiser and Benner, CO:COLLECTION_SUMMARY 2012) to analyze individual amino acids, nucleoside, and carbohydrate content of CO:COLLECTION_SUMMARY the insoluble polymers utilizing MTBSTFA derivatization for insoluble amino CO:COLLECTION_SUMMARY acids. Of the soluble phases, 10 µL were removed from each sample and pooled to CO:COLLECTION_SUMMARY create a QC sample, mixed, and aliquoted into thirteen vials. A QC sample was CO:COLLECTION_SUMMARY run after every sixth injection. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY Synechocystis sp. PCC 6803 [N-1] (ATCC 27184, NCBI Taxonomy ID: 1080229) was TR:TREATMENT_SUMMARY utilized for all experiments. A light-emitting diode photobioreactor (LED PBR) TR:TREATMENT_SUMMARY was engineered to provide a rectified sinusoidal waveform light profile which TR:TREATMENT_SUMMARY (results in the negative half-cycle being set to zero) via two custom 4000K TR:TREATMENT_SUMMARY White LED panels (Reliance Laboratories, Port Townsend WA) arranged opposite a TR:TREATMENT_SUMMARY water bath facing inwards, 5% CO2 at 200 mL min-1 via in-house gas mixing and TR:TREATMENT_SUMMARY custom aerators to provide sufficient mixing, 27-30°C temperature control via a TR:TREATMENT_SUMMARY Huber Ministat and custom water bath (Midwest Custom Aquarium, Starbuck MN), and TR:TREATMENT_SUMMARY improved light penetration at high volumes via custom flat-panel reactors (FPRs) TR:TREATMENT_SUMMARY built in a circular geometry to maximize mixing (Allen Scientific Glass, Boulder TR:TREATMENT_SUMMARY CO) (Figure S1). At the peak, 1,600 ?mol photons m-2s-1 (?E) was provided as TR:TREATMENT_SUMMARY measured by LightScout Quantum Meter (Model: 3415FXSE). . A single LED-PBR was TR:TREATMENT_SUMMARY inoculated and entrained to sinLD cycles for two days; this entrained culture TR:TREATMENT_SUMMARY was then use inoculated three biological triplicate FPRs in the LED PBR (Figure TR:TREATMENT_SUMMARY S2). Reactors were cultivated under the sinLD cycle profile for an additional TR:TREATMENT_SUMMARY day of entrainment prior to sampling (total of 3 days of entrainment). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, 6 mL of 75% methanol (MeOH) was added to pellets, vortexed, and SP:SAMPLEPREP_SUMMARY transferred to glass vials. 9 mL of 100% methyl tert-butyl ether (MTBE) was SP:SAMPLEPREP_SUMMARY added, vortexed for 30 seconds, placed on automatic shaker for 1.5 hours at 4 SP:SAMPLEPREP_SUMMARY ºC, and sonicated for 15 minutes. 3.75 mL of water was added, each extraction SP:SAMPLEPREP_SUMMARY was vortexed by hand for 1 minute, and centrifuged for 10 minutes at 3,270g at SP:SAMPLEPREP_SUMMARY 4ºC. A biphasic solution with a pellet formed: the top, green MTBE layer and SP:SAMPLEPREP_SUMMARY the bottom, clear MeOH:H2O layer were separated into separate tubes and dried SP:SAMPLEPREP_SUMMARY under N2,gas overnight. The pellet was stored at -80 ºC. After drying, the MTBE SP:SAMPLEPREP_SUMMARY layer was resuspended in 100 uL 1:1 toluene:MeOH, transferred to a LC-MS vial SP:SAMPLEPREP_SUMMARY insert, and stored at -80C for <1 month prior to MS analysis. The MeOH:H2O layer SP:SAMPLEPREP_SUMMARY was resuspended in 1 mL of 1:1 H2O:MeOH, transferred to a 1.7 mL centrifuge tube SP:SAMPLEPREP_SUMMARY and spun at 15,000g for 2 minutes at 4 ºC. The supernatant was split into two SP:SAMPLEPREP_SUMMARY 465 µL aliquots—one for GCMS and one for LC(HILIC)MS—in glass vials and SP:SAMPLEPREP_SUMMARY dried under N2,gas. The protocol outlined above is suitable for filter-quenched SP:SAMPLEPREP_SUMMARY cyanobacteria samples and centrifuged cell pellets. The polar methanol/water SP:SAMPLEPREP_SUMMARY fraction resulting from the biphasic extraction was processed for analysis by SP:SAMPLEPREP_SUMMARY hydrophilic interaction liquid chromatography (HILIC) LC-MS. Dried samples were SP:SAMPLEPREP_SUMMARY resuspended in 100 µL 1:1 H2O:MeOH and 10 µL were aliquoted into a pooled QC SP:SAMPLEPREP_SUMMARY sample. Samples were stored at -80 ºC until analysis. The pooled QC sample was SP:SAMPLEPREP_SUMMARY mixed and aliquoted into twelve vials. A QC injection was run every tenth SP:SAMPLEPREP_SUMMARY injection. The dried polar fraction for analysis by GC-MS was stored at -80 ºC SP:SAMPLEPREP_SUMMARY until derivatization, immediately prior to MS analysis. Samples were derivatized SP:SAMPLEPREP_SUMMARY in 30 uL methoxyamine HCl and 30 uL MSTFA, as specified in the following SP:SAMPLEPREP_SUMMARY section. Ten microliters were removed from each sample to create a pooled QC SP:SAMPLEPREP_SUMMARY sample, mixed, and aliquoted into thirteen vials. A QC sample was run after SP:SAMPLEPREP_SUMMARY every sixth injection. The non-polar MTBE phase was processed for non-targeted SP:SAMPLEPREP_SUMMARY LC-MS analysis. Twenty microliters from each sample were pooled, mixed, and SP:SAMPLEPREP_SUMMARY aliquoted into thirteen pooled QC samples. QC injections were placed after every SP:SAMPLEPREP_SUMMARY sixth injection. An acid hydrolysis protocol was developed for identification of SP:SAMPLEPREP_SUMMARY amino acids, nucleosides, and carbohydrates bound in insoluble pellet of SP:SAMPLEPREP_SUMMARY protein, DNA/RNA, and polysaccharides, respectively. Pellets remaining from the SP:SAMPLEPREP_SUMMARY biphasic extraction were removed from storage at -80 degrees C and residual SP:SAMPLEPREP_SUMMARY solvent was evaporated under nitrogen gas. Pellets were re-suspended in 3 mL of SP:SAMPLEPREP_SUMMARY 6 M hydrochloric acid (HCl) using vigorous vortexing and pipette re-suspension. SP:SAMPLEPREP_SUMMARY The resulting suspension was a bright teal. The suspension was transferred SP:SAMPLEPREP_SUMMARY equally two three separate glass vials for hydrolysis of the separate polymer SP:SAMPLEPREP_SUMMARY constituents. Hydrolysis of proteins to amino acids was completed with a SP:SAMPLEPREP_SUMMARY hydrochloric acid (HCl) hydrolysis, based on previously published protocols SP:SAMPLEPREP_SUMMARY (Fountoulakis and Lahm 1998). Briefly, vials were incubated at 110 degrees C for SP:SAMPLEPREP_SUMMARY with a loose cap seal. After 4 hours, the acid in each vial was entirely SP:SAMPLEPREP_SUMMARY evaporated; 1 mL of 6 M HCl was added to each vial, vortexed, sealed tightly, SP:SAMPLEPREP_SUMMARY and returned to 110 degrees C. After a total of 24 hours, vials were removed, SP:SAMPLEPREP_SUMMARY and remaining acid was evaporated under nitrogen gas. Samples were resuspended SP:SAMPLEPREP_SUMMARY in 150 µL of 1:1 MeOH:H2O, 20 uL was removed to create a pooled QC sample, the SP:SAMPLEPREP_SUMMARY pooled QC was aliquoted into fourteen vials, and the solvent was evaporated SP:SAMPLEPREP_SUMMARY under nitrogen gas. Amino acid samples were derivatized in 30 uL of methoxyamine SP:SAMPLEPREP_SUMMARY HCl in pyridine and 30 uL of MTBSTFSA. The peak integration of each amino acid SP:SAMPLEPREP_SUMMARY in each sample was manually checked and curated in the software Chromeleon™. SP:SAMPLEPREP_SUMMARY Aspartic acid with 2- and 3-derivitization agent modifications were detected and SP:SAMPLEPREP_SUMMARY summed for the total spectral abundance. Threonine with 2- and 3-derivitization SP:SAMPLEPREP_SUMMARY agent modifications were detected and summed for the total spectral abundance. SP:SAMPLEPREP_SUMMARY Polysaccharides and nucleic acid polymers were hydrolyzed to nucleosides using a SP:SAMPLEPREP_SUMMARY modified protocol from Huang et al. (2012) (Huang, Kaiser, and Benner 2012). SP:SAMPLEPREP_SUMMARY Briefly, vials were incubated at 130 degrees C for 10 minutes, removed and SP:SAMPLEPREP_SUMMARY allowed to cool, and 100 uL was transferred to a glass teardrop vial. The SP:SAMPLEPREP_SUMMARY remaining pellet was incubated at 160 degrees C for 40 minutes, removed and SP:SAMPLEPREP_SUMMARY allowed to cool, re-suspended in 100 uL LC-MS grade water, vortexed for 15 SP:SAMPLEPREP_SUMMARY seconds, centrifuged at 1,500g for 2 minutes, and the supernatant was SP:SAMPLEPREP_SUMMARY transferred to the glass teardrop vial which contained purines from the 130 SP:SAMPLEPREP_SUMMARY degree C incubation. The acid was evaporated under nitrogen gas. Samples were SP:SAMPLEPREP_SUMMARY removed after the 10 minute 130ºC incubation to preserve purines (guanine and SP:SAMPLEPREP_SUMMARY adenine) from degradation during the 40 minute 160ºC incubation. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For non-targeted GC-MS experiments, metabolites were detected using a Trace 1310 CH:CHROMATOGRAPHY_SUMMARY GC coupled to a Thermo ISQ mass spectrometer. Samples (1 µL) were injected at a CH:CHROMATOGRAPHY_SUMMARY 10:1 split ratio to a 30 m TG-5MS column (Thermo Scientific, 0.25 mm i.d., 0.25 CH:CHROMATOGRAPHY_SUMMARY ?m film thickness) with a 1.2 mL/min helium gas flow rate. GC inlet was held at CH:CHROMATOGRAPHY_SUMMARY 285°C. The oven program started at 140°C for 1 min, followed by a ramp of CH:CHROMATOGRAPHY_SUMMARY 15°C/min to 330°C, and 5 min hold. Masses between 50-650 m/z were scanned at 5 CH:CHROMATOGRAPHY_SUMMARY scans/sec under electron impact ionization. Transfer line and ion source were CH:CHROMATOGRAPHY_SUMMARY held at 300 and 260°C, respectively. Pooled QC samples were injected after CH:CHROMATOGRAPHY_SUMMARY every 6 actual samples. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Thermo ISQ CH:COLUMN_NAME Trace 1310 GC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo ISQ MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Raw data was converted to *.CSV with Waters® Databridge. For idMS/MS (RP-LC-MS MS:MS_COMMENTS runs), a file was converted for low-collision, high-collision, and LockSpray for MS:MS_COMMENTS each sample. Peaks were detected within the XCMS workflow using the Centwave MS:MS_COMMENTS algorithm (Smith et al. 2006). MS:MS_RESULTS_FILE ST001196_AN001992_Results.txt UNITS:spectral abundance per cell Has m/z:No Has RT:No RT units:No RT data #END