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. 2018 Nov 16;9:2725. doi: 10.3389/fmicb.2018.02725

FIGURE 6.

FIGURE 6

LC-MS analysis from recycling mutants, lacking MurP transporter. S. aureus wild type (wt) cells, a murP transporter mutant (murP::Tn) and a murQ-operon mutant (ΔmupGmurQPR) were grown to stationary phase (24 h) in LB. Bacterial cultures were centrifuged and 1 ml of each supernatant was transferred to 500 μl of ice-cold acetone to remove macromolecules and proteins from the samples. After centrifugation, culture supernatants were dryed in the speedvac, solved in 50 μl of Millipore water and 2 μl of the samples were injected to the HPLC column. MS analysis of the culture supernatants was performed in positive-ion mode and mass spectra are presented as extracted ion chromatograms (EIC × 105 counts per second [cps]) in orange. In the culture supernatants of both mutants, an additional compound accumulates (murP::Tn, observed mass of (M+H)+ = 497.197 m/z; ΔmupGmurQPR, observed mass of (M+H)+ = 497.195 m/z). The mass of this compound in the supernatant corresponds to a disaccharide made of GlcNAc and MurNAc. This compound was further analyzed by MS fragmentation and identified as MurNAc-GlcNAc (see Supplementary Figure 5). MS data are shown as representative from three biological replicates.