The peptidoglycan cross-bridges of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium consist of the sequences Gly(5), l-Ala(2), and d-Asx, respectively. Expression of the fmhB, femA, and femB genes of S. aureus in E. faecalis led to the production of peptidoglycan precursors substituted by mosaic side chains that were efficiently used by the penicillin-binding proteins for cross-bridge formation. The Fem transferases were specific for incorporation of glycyl residues at defined positions of the side chains in the absence of any additional S. aureus factors such as tRNAs used for amino acid activation. The PBPs of E. faecalis displayed a broad substrate specificity because mosaic side chains containing from 1 to 5 residues and Gly instead of l-Ala at the N-terminal position were used for peptidoglycan cross-linking. Low affinity PBP2a of S. aureus conferred beta-lactam resistance in E. faecalis and E. faecium, thereby indicating that there was no barrier to heterospecific expression of resistance caused by variations in the structure of peptidoglycan precursors. Thus, conservation of the structure of the peptidoglycan cross-bridges in members of the same species reflects the high specificity of the enzymes for side chain synthesis, although this is not essential for the activity of the PBPs.