Fig. 3. MurJ activity is required for ColM-dependent cleavage of lipid II in spheroplasts.

Cells lacking the ColM receptor FhuA and producing the indicated MurJ variants were grown, labeled, and treated with MTSES as for figure 1. Spheroplasts were then prepared. In all but one case, spheroplasts were pelleted and resuspended in ColM reaction buffer with sucrose and added MTSES (0.8 mM) as indicated. The Lysis + sample was resuspended in buffer lacking sucrose to lyse the spheroplasts. ColM (100 µg) was added to the prepared spheroplasts as indicated and they were incubated for 15 min at 37°C. Lipid intermediates were detected after butanol extraction by scintillation counting. Statistics are as for figure 1.