Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In Staphylococcus aureus, pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into cross-bridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine cross-bridge. FmhA incorporates serine residues at positions 3 and 5 of the cross-bridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets penicillin-binding proteins, in both methicillin-sensitive and methicillin-resistant strains of S. aureus. FmhC is encoded by a gene immediately adjacent to lytN, which specifies a hydrolase that cleaves the bond between the fifth glycine of cross-bridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC. Together, these observations suggest that FmhA and FmhC generate peptidoglycan cross-bridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.
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