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. 2023 Oct 23;14:6694. doi: 10.1038/s41467-023-42478-x

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Allelic replacement at the plsM locus of Msmg mutants rescued with plsC_coli was analyzed as in Fig. 1b. b Growth characteristics of WT Msmg mc²155 (black circles), MsmgΔplsM/pSETetR-plsMtb (black triangles), and MsmgΔplsM/pMVGH1-plsC_coli (clones # 32 and 34; light and dark green triangles, respectively) in 7H9-ADC-0.05% tyloxapol at 37 °C (in the presence of 50 ng/mL ATc for MsmgΔplsM/pSETetR-plsMtb). Shown are the means ± SD of three independent growth curves (n = 3 independent biological triplicate) for each strain. c De novo phospholipid synthesis in WT Msmg mc²155, MsmgΔplsM/pMVGH1-plsC_coli (clones # 32 and 34) and MsmgΔplsM/pSETetR-plsMtb grown in 7H9-ADC-0.05% tyloxapol at 37 °C (in the presence of 50 ng/mL ATc for MsmgΔplsM/pSETetR-plsMtb). [1,2-¹⁴C]acetic acid was added to bacterial cultures when they reached an OD₆₀₀ nm of 0.8 after which cultures were incubated for another 8 h at 37 °C with shaking. [1,2-¹⁴C]acetic acid-derived lipids were analyzed by TLC in the solvent system CHCl₃:CH₃OH:H₂O (70:20:2 by vol.). The same total counts (dpm) were loaded per lane. Ac₂PIM₂ are tetraacylated forms of phosphatidyl-myo-inositol dimannosides. Other lipid abbreviations are as in Fig. 1. The results shown are representative of two independent experiments. d Fatty acid composition of WT Msmg mc²155 and MsmgΔplsM/pMVGH1-plsC_coli (two different clones, # 32 and # 34; dark and light green bars, respectively) grown in 7H9-ADC-0.05% tyloxapol at 37 °C (same medium as used in panels b and c). C19:0: tuberculostearic acid. e Extracted ion chromatograms (EICs) showing the lysophosphatidic acid (LPA) products resulting from the incubation of membranes prepared from Msmg/pMVGH1 and MsmgΔplsM/pMVGH1-plsC_coli (clone # 32) with [¹³C]-G3P and C16:0-CoA. sn-2 palmitoyl transferase activity is dominant in Msmg/pMVGH1 whereas it is barely detectable in MsmgΔplsM/pMVGH1-plsC_coli. C16:0 in the latter strain is essentially transferred to position sn-1 of G3P. Source data for panels (b and d) are provided as a Source Data file.