Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with To mount a successful infection, Gram-positive pathogens display proteins on the bacterial surface that adhere to specific receptors on host tissues or provide for microbial escape from the host's immune response (1). Protein display on the bacterial surface involves the covalent linkage of polypeptides to the cell wall envelope (2). As reported for protein A of Staphylococcus aureus, surface proteins are synthesized as P1 precursor molecules in the bacterial cytoplasm, bearing an N-terminal signal peptide and a C-terminal sorting signal (3). The 35-residue sorting signal is composed of a LPXTG motif, a hydrophobic domain, and a tail of positively charged residues (4). After translocation across the cytoplasmic membrane, the N-terminal signal peptide is removed by signal peptidase, thereby generating the P2 precursor (4). The C-terminal sorting signal retains the P2 precursor species within the secretory pathway and permits substrate recognition at the LPXTG motif (4, 5). Sortase, a membrane-anchored transpeptidase, cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif (6, 7). Cleaved polypeptides are initially tethered as thioester-linked intermediates to the active site sulfhydryl residue of sortase enzymes (8). Nucleophilic attack of the amino group of pentaglycine cross-bridges within the staphylococcal peptidoglycan resolves this acyl-enzyme intermediate (8), resulting in the formation of an amide bond that tethers the C terminus of surface protein to the cell wall peptidoglycan (9 -13).The peptidoglycan of S. aureus is synthesized in three cellular compartments, the cytoplasm, the membrane and the cell wall envelope ( (18,19). Lipid II is translocated across the cytoplasmic membrane and functions as a substrate for two cell wall biosynthetic reactions that require mono-or bifunctional transglycosylases and transpeptidases (20). In the transglycosylation reaction, lipid II is polymerized to generate linear peptidoglycan strands with the repeating disaccharide (MurNAc-GlcNAc) n . This reaction is fueled by the hydrolysis of lipid II and by further hydrolysis of the undecaprenolpyrophosphate product, which is translocated across the plasma membrane into the cytoplasm (21). Linear peptidoglycan strand...