Lipidomic assays for the effects of StpK7, MSMEG_1198, MSMEG_1197, and MSMEG_1195 on mycobacterial lipid homeostasis in Mycobacterium smegmatis

To investigate the effects of MSMEG_1195, MSMEG_1197, MSMEG_1198, and StpK7 (MSMEG_1200) on the lipid homeostasis of <em>M. smegmatis</em>, we constructed <em>stpK7</em> knockout strains through homologous recombination and overexpression strains of <em>MSMEG_1195</em>, <em>MSMEG_1197</em>, and <em>MSMEG_1198</em> using the pMV261 plasmid. These strains were cultured in 100 mL 7H9 medium containing 25 µg/mL kanamycin to an OD600 of 1.0, and then harvested for lipidomic assays. Lipid extraction, ultra-performance liquid chromatography, mass spectrometry, and lipid structural analyses were conducted by Novogene (Beijing, China). For detailed procedures regarding lipid extraction and mass spectrometry detection, please refer to previous literature (https://doi.org/10.1194/jlr.D700041-JLR200). Metabolite identification was accomplished using databases such as NIST (https://www.nist.gov/), Lipidmaps (http://www.lipidmaps.org), and Mtb LipidDB (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073466/). Each sample was replicated independently at least five times, and univariate analysis (t-test) was employed to calculate the statistical significance (P value) and fold change of the metabolites between the means of the two groups. The abbreviations WT and Ko indicate the wild-type strain (WT/pMV261) and the <em>stpK7</em>-knockout strain (Ko<em>stpK7</em>/pMV261), respectively; while OE95, OE97, and OE83 separately represent the <em>MSMEG_1195</em>-overexpressed, <em>MSMEG_1197</em>-overexpressed, and <em>MSMEG_1198</em>-overexpressed strains (abbreviated as OE-<em>MSMEG_1195</em>, OE-<em>MSMEG_1197</em>, and OE-<em>MSMEG_1198</em>).