Abstract
The surfaces of most Gram-positive bacterial cells, including that of Staphylococcus aureus (S. aureus), are heavily decorated with proteins that coordinate cellular interactions with the extracellular space. In S. aureus, sortase A is the principal enzyme responsible for covalently anchoring proteins, which display the sorting signal LPXTG, onto the peptidoglycan (PG) matrix. Considerable efforts have been made to understand the role of this signal peptide in the sortase-mediated reaction. In contrast, much less is known about how the primary structure of the other substrate involved in the reaction (PG stem peptide) could impact sortase activity. To assess the sortase activity, a library of synthetic analogs of the stem peptide that mimic naturally existing variations found in the S. aureus PG primary sequence were evaluated. Using a combination of two unique assays, we showed that there is broad tolerability of substrate variations that are effectively processed by sortase A. While some of these stem peptide derivatives are naturally found in mature PG, they are not known to be present in the PG precursor, lipid II. These results suggest that sortase A could process both lipid II and mature PG as acyl-acceptor strands that might reside near the membrane, which has not been previously described.