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. Author manuscript; available in PMC: 2020 May 8.
Published in final edited form as: Cell Host Microbe. 2019 May 8;25(5):668–680.e7. doi: 10.1016/j.chom.2019.04.002

Figure 2: Genetic deletion of the serine palmitoyltransferase enzyme in B. thetaiotaomicron results in sphingolipid-deficient bacteria.

Figure 2:

(A) Schematic of sphingolipid biosynthesis, in which serine palmitoyltransferase (SPT) catalyzes the addition of amino acids (e.g. serine) to acyl-coenzyme A (CoA) to form an N-acylated sphingoid backbone (e.g. 3-ketosphinganine). (B) Representative TLC plate verifying that ceramide phosphoethanolamine (CerPE) in wild type B. thetaiotaomicron (BTWT) is absent in the B. theta spt mutant (BTΔSPT). CerPE (Rf 0.35) is shown after ninhydrin staining (red) on TLC plates below the phosphorylethanolamine (PE) band and run with a CerPE standard, C12 sphingosyl-PE (C12 SPE). (C) Representative tick plots of the elution peak at the CerPE (679 m/z) retention time (RT) on a C8-column (7.7 min). The CerPE sphingolipid peak present in BTWT lipids (blue box) was absent in BTΔSPT lipids (red box). Data is representative of 3 biological replicates. (D) Isolation of purified CerPE from B. thetaiotaomicron was confirmed using LC-MS/MS. Tail lengths corresponding to the known masses of CerPE (679, 693, and 707m/z) were identified. Structures are shown above. (E) Representative TLC plate stained with ninhydrin (red) showing the CerPE band (Rf 0.35) is present in BTWT and SPT-complemented BTΔSPT strains. (F) Log2 fold-change (FC) difference between each B. thetaiotaomicron gene significantly changed (>2-fold upregulated, FDR<0.01) in the wild-type or mutant strain after growth in minimal and rich media. Genes upregulated in BTWT relative to BTΔSPT are in red and downregulated are in blue. Annotated gene functions, if available, are listed on the right. N=3. Only genes significantly changed in both rich and minimal media are displayed. See also Figure S2, Table S1, and Data File S2.