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TLC analysis of glycolipids in total membrane lipid extracts from E. faecium . Glycolipid profiles of E. faecium SM1, SM1 lafB T577C, SM1Δ lafB , and SM1 Δ lafB /pAT28:: lafB mutants were compared to WT E. faecium SM1. Purified lipid standards included Gal₂DAG [1,2-diacyl-3-O-(α-D-galactosyl(1→6)-β-D-galactosyl)-sn-glycerol] and Glc ₁ DAG [1,2-diacyl-3-O-(α-D-glucopyranosyl)-sn-glycerol] (Avanti Polar Lipids). Glycolipid species Glc ₁ DAG, Glc ₂ DAG, and Gal ₂ DAG were identified based on migration relative to standards. Additional bands representing unknown lipid species are also indicated. The absence of glycolipid bands in the Δ lafB mutant and their restoration in the complemented strain demonstrates the role of lafB in glycolipid biosynthesis. Data are representative of three independent experiments.

Journal: mBio

Article Title: Loss of LafB activity reverses daptomycin resistance in E. faecium

doi: 10.1128/mbio.00715-25

Figure Lengend Snippet: TLC analysis of glycolipids in total membrane lipid extracts from E. faecium . Glycolipid profiles of E. faecium SM1, SM1 lafB T577C, SM1Δ lafB , and SM1 Δ lafB /pAT28:: lafB mutants were compared to WT E. faecium SM1. Purified lipid standards included Gal₂DAG [1,2-diacyl-3-O-(α-D-galactosyl(1→6)-β-D-galactosyl)-sn-glycerol] and Glc ₁ DAG [1,2-diacyl-3-O-(α-D-glucopyranosyl)-sn-glycerol] (Avanti Polar Lipids). Glycolipid species Glc ₁ DAG, Glc ₂ DAG, and Gal ₂ DAG were identified based on migration relative to standards. Additional bands representing unknown lipid species are also indicated. The absence of glycolipid bands in the Δ lafB mutant and their restoration in the complemented strain demonstrates the role of lafB in glycolipid biosynthesis. Data are representative of three independent experiments.

Article Snippet: Approximately 5 μL of each of the two standards (Gal 2 DAG: 1,2-diacyl-3- O -(α- d -galactosyl1-6)-β- d -galactosyl- sn -glycerol Avanti Polar Lipids 840524P, Glc 1 DAG: 1,2-diacyl-3-O-(α-D-glucopyranosyl)-sn-glycerol Avanti Polar Lipids 840522P) and ~15 μL of each sample was spotted on the baseline of a Silica gel 60 TLC plate.

Techniques: Membrane, Purification, Migration, Mutagenesis