REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant

The peptidoglycan of Gram-positive bacteria consists of glycan chains with attached short peptide stems cross-linked to one another by glycyl bridges. The bridge of Staphylococcus aureus has five glycyl units and that of its FemA mutant has one. These long- and short-bridge cross-links create totally different cell-wall architectures. S. aureus and its FemA mutant grown in the presence of an alanine-racemase inhibitor were labeled with d-[1-13C]alanine, l-[3-13C]alanine, [2-13C]glycine, and l-[5-19F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between 13C-labeled alanines and 19F-labeled lysine incorporated in the peptidoglycan. The alanyl 13C labels in the parent strain were preselected for C{F} and C{P} REDOR measurement by their proximity to the glycine label using 13C13C spin diffusion. The observed 13C13C and 13C31P distances are consistent with a tightly packed architecture containing only parallel stems in a repeating structural motif within the peptidoglycan. Dante selection of d-alanine and l-alanine frequencies followed by 13C13C spin diffusion rules out scrambling of carbon labels. Cell walls of FemA were also labeled by a combination of d-[1-13C]alanine and l-[15N]alanine. Proximity of chains was measured by C{N} and N{C} REDOR distances and asymptotic plateaus, and both were consistent with a mixed-geometry model. Binding of an 19F-labeled eremomycin analog in the FemA cell wall matches that of binding to the parent-strain cell wall and reveals the proximity of parallel stems in the alternating parallel–perpendicular mixed-geometry model for the FemA peptidoglycan lattice. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

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