Cytoplasmic peptidoglycan intermediate levels in Staphylococcus aureus.

Abstract

Intracellular cytoplasmic peptidoglycan (PG) intermediate levels were determined in Staphylococcus aureus during log-phase growth in enriched media. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode, and amine intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode. Levels of UDP-linked intermediates in S.aureus varied from 1.4μM for UDP-GlcNAc-Enolpyruvyate to 1200μM for UDP-MurNAc. Levels of amine intermediates (L-Ala, D-Ala, D-Ala-D-Ala, L-Glu, D-Glu, and L-Lys) varied over a range of from 860μM for D-Ala-D-Ala to 30-260mM for the others. Total PG was determined from the D-Glu content of isolated PG, and used to estimate the rate of PG synthesis (in terms of cytoplasmic metabolite flux) as 690μM/min. The total UDP-linked intermediates pool (2490μM) is therefore sufficient to sustain growth for 3.6min. Comparison of UDP-linked metabolite levels with published pathway enzyme characteristics demonstrates that enzymes on the UDP-branch range from >80% saturation for MurA, Z, and C, to <5% saturation for MurB. Metabolite levels were compared with literature values for Escherichia coli, with the major difference in UDP-intermediates being the level of UDP-MurNAc, which was high in S.aureus (1200μM) and low in E.coli (45μM).