MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/m_MTBLS11404_LC-MS_alternating_hilic_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/m_MTBLS11404_LC-MS_alternating_reverse_phase_lipid_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/a_MTBLS11404_LC-MS_alternating_hilic.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/a_MTBLS11404_LC-MS_alternating_reverse-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/s_MTBLS11404.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S05_Exp_1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_20231003041615.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_6_DG_6_Transition_2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S12_Tya_4.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_4_DG_4_Exponential_phase_4.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S09_Tya_1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S07_Exp_3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_5_DG_5_Transition_1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S03_STA_3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_20231003012653.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_3_20220916135418.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_3_DG_3_Exponential_phase_3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S08_Exp_4.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S11_Tya_3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_9_DG_9_Stationary_phase_1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S10_Tya_2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_1_20220916124617.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S04_STA_4.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_MSMS_neg_20231003052403.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_8_DG_8_Transition_4.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_2_20220916132020.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_2_DG_2_Exponential_phase_2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S02_STA_2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_MSMS_neg.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_10_DG_10_Stationary_phase_2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S06_Exp_2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S13_Bnk_1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_11_DG_11_Stationary_phase_3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_MSMS_pos_20231003045010.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_1_DG_1_Exponential_phase_1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/S01_STA_1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_12_DG_12_Stationary_phase_4.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_MSMS_pos.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_4_20220916142814.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/PQC_20231002223729.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404/FILES/RAW_FILES/Sample_7_DG_7_Transition_3.rawprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS11404For accurate metabolite identification, a standard library of ~500 metabolites were analysed before sample testing and accurate retention time for each standard was recorded. This standard library also forms the basis of a retention time prediction model used to provide putative identification of metabolites not contained within the standard library. Acquired LC-MS/MS data was processed in an untargeted fashion using open source software IDEOM v21, which initially used msConvert (ProteoWizard) to convert raw LC-MS files to mzXML format and XCMS to pick peaks to convert to .peakML files. Mzmatch was subsequently used for sample alignment and filtering. IDEOM v21 was utilised for further data pre-processing, organisation and quality evaluation. MetaboLightsPublicLiquid Chromatography MS - alternating - hilicLiquid Chromatography MS - alternating - reverse-phaseThe chromatography utilized a ZIC-p(HILIC) column 5 µm 150 x 4.6 mm with a 20 x 2.1 mm ZIC-pHILIC guard column (both Merck Millipore, Australia) (25 °C). A gradient elution of 20 mM ammonium carbonate (A) and acetonitrile (B) (linear gradient time-%B: 0 min-80%, 15 min-50%, 18 min-5%, 21 min-5%, 24 min-80%, 32 min-80%) was utilised. Flow rate was maintained at 300 μL/min. Samples were kept in the autosampler (6°C) and 10 μL was injected for analysis.An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage.David GillettChristopher K BarlowMonash UniversityRice Universitywhole cell lysate formatmass spectrometry assayPellets were resuspend thoroughly in 200 µL extraction solvent (2:6:1 chloroform:methanol:water v/v/v and spiked with 2 µM generic internal standard (CHAPS/CAPS/PIPES and Tris) at 4 °C, and subject to three freeze-thaw cycles by snap freezing in liquid N2 and then thawing on ice. Samples were mixed on a vibrating mixer (PCV-3000, 1500 RMP, 1 sec, Vortex = Hard, 10, cycle = 60, stop) at 4 °C and then centrifuged at 20,000 ×g, 10 min, 4 °C). 180 µL of supernatant was transferred into new 1.5 mL tube and frozen at -80 °C. Immediately prior to LC-MS analysis samples were thawed on ice and centrifuged at 20,000 ×g, before being transferred to sample vial inserts. Mycobacterium smegmatis str. MC2 155https://www.ebi.ac.uk/metabolights/MTBLS11404Perran Cook.Han-Chung Lee.Ralf Schittenhelm.Thilini Koralegedara.Wei Wong.Debnath Ghosal.Tess Hutchinson.Christopher Barlow.Manasi Mudaliyar.David Gillett. Rice University. gillettdavid948@gmail.com.Chris Greening.Thomas Watts.Joel Steele.Luis Jimenez.Erwin Tanuwidjaya.Nadeesha Athukorala.Ning Hall.Iresha Hanchapola.Jake Locop.Rhys Grinter.After metabolite identification and filtering, samples were normalised by median peak ion intensity, subject to log10 transformation and Pareto scaling in MetaboAnalyst (www.metaboanalyst.ca/): No data filtering was performed (<5000 features). Growth phasechris.barlow@monash.edugillettdavid948@gmail.comSealed cultures of M. smegmatis were harvested during exponential phase (EXP; O2 = 13%, OD600 = ~1.0), the hypoxic transition (TR; O2 = 0.5%, OD600 = 2.6, 12 hours post ODmax) and hypoxic stationary phase (ST; O2 = 0%, OD600 = 2.2) in quadruplicate. 15 mL of culture were pelleted via centrifugation (4,500 ×g, 15 min, 4°C) and washed once via resuspension in 15 mL of 1X PBS, pelleting via centrifugation as before, discarding supernatant and flash freezing the pellet in liquid N2. Pellets were stored at -80C. Metabolomicsintracellular storage of carbonuntargeted analysisuntargeted metabolitesLipidomicsexperimental blankMycobacterium smegmatis str. MC2 155whole cell lysate formatThermo Scientific Dionex Ultimate 3000 HPLC systemThermo Scientific Q Exactive Plusintracellular storage of carbonuntargeted analysisLipidomicsuntargeted metabolitesexperimental blankMycobacterium smegmatis str. MC2 155whole cell lysate formatThermo Scientific Dionex Ultimate 3000 HPLC systemThermo Scientific Q Exactive PlusMS was performed using a Q-Exactive Plus Orbitrap MS (Thermo) at 70,000 resolution operating in rapid switching positive (4 kV) and negative (−3.5 kV) mode electrospray ionization (capillary temperature 300°C; sheath gas flow rate 50; auxiliary gas flow rate 20; sweep gas 2; probe temp 120 °C).falseAn obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storageIn soil ecosystems, obligately aerobic bacteria survive oxygen deprivation (hypoxia) by entering non-replicative persistent states. Little is known about how these bacteria rewire their metabolism to stay viable during persistence. The model obligate aerobe Mycobacterium smegmatis maintains redox homeostasis during hypoxia by mediating fermentative hydrogen production. However, the fate of organic carbon during fermentation, and the associated remodeling of carbon metabolism, is unresolved. Here we systematically profiled the metabolism of M. smegmatis during aerobic growth, hypoxic persistence, and the transition between these states. Using differential isotope labelling, and paired metabolomics and proteomics, we observed rerouting of central carbon metabolism through the pentose phosphate pathway during hypoxia. In addition, we found that M. smegmatis excretes high concentrations of acetate during hypoxia, likely for ATP synthesis and as a carbon overflow mechanism. We show that M. smegmatis excretes high levels of hydrogen and acetate concurrently with upregulating triacylglyceride synthases and accumulating glycerides as carbon stores. Using electron cryotomography (cryo-ET), we observed the presence of large spheroid structures consistent with the appearance of lipid droplets. Thus, in contrast to obligately and facultative anaerobic fermentative bacteria that primarily excrete organic carbon during hypoxia, M. smegmatis also stores this carbon. This novel hybrid metabolism likely provides a competitive advantage in resource-variable environments by allowing M. smegmatis to simultaneously dispose excess reductant during hypoxia and maintain carbon stores to rapidly resume growth upon reoxygenation. Similar strategies may be widely employed by other obligate aerobes throughout resource-variable environments.2026-04-222026-04-22MTBLS11404