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. 2020 Jan 23;295(10):3347–3361. doi: 10.1074/jbc.REV119.010155

Figure 5.

Figure 5.

Making and labeling peptidoglycan substrates to characterize cell wall enzymes. A, accumulation and extraction of Lipid II. Bacterial cultures are treated with an antibiotic that inhibits peptidoglycan synthesis to accumulate Lipid II in cells. The cells are spun down and resuspended in chloroform/methanol for the first extraction, which produces three layers. Lipid II is enriched in a thick interface fraction, whereas the majority of cellular phospholipids partition to the organic layer. A second extraction results in partitioning of Lipid II into the organic phase and UDP-MurNAc-pentapeptide, which is also present in the interface layer, into the aqueous phase. B, the two-step extraction allows isolation of Lipid II variants from the indicated species. C, uncross-linked glycan strands are synthesized from Lipid II using the monofunctional glycosyltransferase SgtB, which recognizes Lipid II variants with different stem peptides. To make cross-linked peptidoglycan, an appropriate bifunctional aPBP that recognizes the stem peptide is used. The aPBP polymerizes Lipid II and cross-links glycan strands. D, a chemical probe can be incorporated within the glycan backbone or stem peptide of peptidoglycan substrates to visualize and assess reaction of the substrates. i, one strategy to label the stem peptide of Lipid II or glycan strands makes use of the transpeptidase PBPX, which exchanges the terminal d-Ala in the stem peptide for a d-amino acid bearing a detectable tag. ii, another stem peptide labeling strategy selectively couples an amine-reactive probe to the side-chain primary amine at position 3 of the stem peptide. iii, a strategy to label the sugar backbone uses GalT to attach a [14C]galactose radiolabel at the nonreducing end of glycan strands. E, methods to digest peptidoglycan products for structural characterization by LC-MS. i, mutanolysin digestion and NaBH4 reduction of cross-linked peptidoglycan produce muropeptide species. ii, ColM treatment of uncross-linked glycan strands produces delipidated glycan diphosphate products.