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A comparative analysis of mycobacterial lipids using liquid chromatography mass spectrometry-based lipidomics

ABSTRACT: The lipid content of twenty different species across the genus Mycobacterium was analysed by LC-MS. Single colonies were inoculated into Middlebrook 7H9 medium and grown to an OD 600nm of one. One mL of culture was transferred onto a filter and filters were placed on 7H10 plates and incubated for 6 doubling times. Lipids were extracted with chloroform: methanol in 2:1, 1:1 and 1:2 ratios. Extracts were pooled and dried and resuspended in LC mobile phase A. Analysis was done using a BETASIL Diol-100 column on an Ultimate 3000 HPLC system attached to a Thermo Q Exactive Orbitrap Mass Spectrometer. Mobile phase A was isopropanol:hexane at 70:30 and mobile phase B was isopropanol: methanol at 70:30, both phases contained 0.02% [m/v] formic acid and 0.01% [m/v] ammonium hydroxide.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mycolicibacter Longobardus (ncbitaxon:1108812) Mycobacterium Tuberculosis H37ra (ncbitaxon:419947) Mycobacterium Smegmatis Str. Mc2 155 (ncbitaxon:246196) Mycolicibacterium Flavescens (ncbitaxon:1776) Mycobacterium Alsense (ncbitaxon:324058) Mycolicibacter Hiberniae (ncbitaxon:29314) Mycobacterium Angelicum (ncbitaxon:470074) Mycolicibacterium Aurum (ncbitaxon:1791) Mycolicibacterium Conceptionense (ncbitaxon:451644) Mycolicibacterium Fallax (ncbitaxon:1793) Mycobacterium Marinum (ncbitaxon:1781) Mycolicibacterium Litorale (ncbitaxon:758802) Mycobacteroides Abscessus (ncbitaxon:36809) Mycolicibacterium Gilvum (ncbitaxon:1804) Mycobacterium Palustre (ncbitaxon:153971) Mycolicibacterium Aromaticivorans (ncbitaxon:318425) Mycobacterium Fragae (ncbitaxon:1260918) Mycobacterium Gordonae (ncbitaxon:1778) Mycolicibacterium Phlei (ncbitaxon:1771) Mycobacterium Branderi (ncbitaxon:43348)

SUBMITTER: Luiz Pedro Carvalho

PROVIDER: MSV000089983 | MassIVE | Tue Jul 26 12:54:00 BST 2022

REPOSITORIES: MassIVE

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Project description:In order to provide information about the gene expression response that occurs when cells experience a change in carbon source, succinate limited chemostat cultures of Methylobacterium extorquens AM1 were grown to and maintained at an OD of ~0.63, transferred to flasks and methanol was added. Cells were harvested for RNA extraction at time: 0 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr and 6 hr post transition. At 30 min, a no methanol addition sample was extracted as a carbon starvation control. These data were used in conjunction with flux, enzymatic and metabolite measurements to assess the changes in central metabolism during this transition. Abstract from manuscript: When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate to methanol growth by the methylotrophic bacterium Methylobacterium extorquens, analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylenetetrahydrofolate dehydrogenase as one control point. This “downstream priming” mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints. Gene expression in succinate limited chemostat cultures was compared to gene expression in cells transferred to flasks before and after methanol addition. As a control, a time = 0 sample (RNA prepared from cells harvested directly from the chemostat) was compared to a time = 0 sample immediately obtained after the cells were transferred to flasks, before methanol was added in order to identify changes due to flask transfer. A carbon starvation control was also done comparing expression from time = 0 (chemostat cells) to cells transferred to flasks for 30 min with no carbon source added. Two biological replicates each with two techinal replicates (dye swap) were analyzed for time = 0 (chemostat) vs 10 min, 30 min, 1 hr and 2 hr after methanol addition. One biological replicate with two technical replicates (dye swap) were analyzed for time = 0 (chemostat) vs time = 0 (flask transfer), and time = 0 (chemostat) vs time = 4 hr, 6 hr and 30 min no methanol addition.

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A comparative analysis of mycobacterial lipids using liquid chromatography mass spectrometry-based lipidomics

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