Figure 6. Muropeptide composition of PG following mecillinam treatment.

Cells of MG1655 [WT] (A) or HC408 [Δslt] (B) were grown to an OD₆₀₀ of approximately 0.5 and diluted 1:20 into fresh LB medium with or without mecillinam (10 μg/ml) as indicated. Growth was continued for an additional 3 hours and PG sacculi were prepared from cells of each culture. The purified PG was then digested with the muramidase mutanolysin and the resulting muropeptides were reduced and analyzed by LC/MS. Total ion count chromatograms are shown with the chromatograms of the mecillinam-treated samples off-set for clarity. Note that the scales of each chromatogram are not identical. They were scaled to show the relative muropeptide composition rather than the total amount of material. For example, the total peak area in the chromatogram from mecillinam-treated WT cells is one third that of the corresponding untreated sample even though an equivalent of twice the volume was injected. Schematics of several major muropeptide products are shown near their corresponding peaks with the numbers referring to the type of crosslink (4-3 v.s. 3-3). The identities for all labeled peaks and the quantification of their relative amounts are listed in Table S1. The results were reproducible over two biological replicates of each sample and three technical replicates for each biological replicate.