Substrate Preferences Establish the Order of Cell Wall Assembly in <i>Staphylococcus aureus</i>

The Gram-positive bacterial cell wall is a large supramolecular structure and its assembly requires coordination of complex biosynthetic pathways. In the step that merges the two major biosynthetic pathways in Staphylococcus aureus cell wall assembly, conserved protein ligases attach wall teichoic acids to peptidoglycan, but the order of biosynthetic events is a longstanding question. Here, we use a chemical approach to define which of the possible peptidoglycan intermediates are substrates for wall-teichoic acid ligases, thereby establishing the order of cell wall assembly. We have developed a strategy to make defined glycan chain-length polymers of either un-cross-linked or cross-linked peptidoglycan, and we find that wall teichoic acid ligases cannot transfer wall teichoic acid precursors to the cross-linked substrates. A 1.9 Å crystal structure of a LytR-CpsA-Psr (LCP) family ligase in complex with a wall teichoic acid precursor defines the location of the peptidoglycan binding site as a long, narrow groove, and suggests that the basis for selectivity is steric exclusion of cross-linked peptidoglycan. Consistent with this hypothesis, we have found that chitin oligomers are good substrates for transfer, showing that LCPs do not discriminate cross-linked from un-cross-linked peptidoglycan substrates by recognizing features of the un-cross-linked stem peptide. We conclude that wall teichoic acids are coupled to un-cross-linked peptidoglycan chains at an early stage of peptidoglycan synthesis and may create marks that define the proper spacing of subsequent cross-links