Abstract
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.
Highlights
Peptidoglycan is a large macromolecule that surrounds bacteria to support cell integrity and shape [1,2,3]
Efforts to elucidate staphylococcal cell wall synthesis in the late 1960s used S. aureus crude enzyme preparations and demonstrated the transfer of glycine from glycyl-tRNA to the peptidoglycan biosynthetic intermediate lipid II [C55-(PO4)2-N-acetylmuramic acid (NAM)(L-Ala-D-iGln-L-Lys-DAla-D-Ala)-NAG] [20,21,22,23,24]. Edman degradation of these reaction products revealed that the cross-bridge is synthesized by the sequential addition of glycine from glycyl-tRNA to the e amino group of L-Lys, demonstrating that peptidyl-transfer occurs at the NH2 terminus in a ribosome-independent manner [25]
Sequence alignments between Fem and Fmh factors of S. aureus strain Newman and lysostaphin immunity factor (Lif) and endopeptidase resistance (Epr) demonstrate that FmhA and FmhC share greater sequence identity with Lif and Epr than FemA or FemB and that FemX is distinct from the other six factors (Fig. 1A)
Summary
Peptidoglycan is a large macromolecule that surrounds bacteria to support cell integrity and shape [1,2,3]. The HPLC elution profiles of mutanolysin digests of peptidoglycan preparations from strain Newman and isogenic variants fmhA and fmhC reveal comparable degrees of low (eluting between 20 and 40 min) and highly cross-linked materials (eluting between 50 and 130 min) (Fig. 1D). FmhA and FmhC incorporate serine into the cross-bridge of S. aureus peptidoglycan
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