Fig 5. Hyperactive mutant PBP2^L61R is more sensitive to mecillinam.

a. Modeled “off state” structure of E. coli PBP2 from H. pylori PBP2 using Phyre 2. Structural information lacks on the juxta-membrane, transmembrane helix and cytoplasmic regions of PBP2. Residue Leu61 is colored in red and shown as spheres. Active site Ser330 is colored in pink and shown as spheres. b. Hyperactive mutant PBP2^L61R interacts similar like PBP2^WT with RodA and MreC. RodA and MreC were fused with mCherry, and PBP2^WT and PBP2^L61R were fused with mKO. c. Phase contrast and fluorescence images of cells expressing PBP2^L61R were less sensitive to A22 but hypersensitive to mecillinam in liquid culture. LMC500 strain expressing nothing, or PBP2^WT, or PBP2^L61R were grown in LB medium at 37°C. IPTG induction (15 μM), and A22 treatment (10 mg·L^-1) or mecillinam treatment (2 mg·L^-1) were applied to each culture for 2 mass doublings. Arrows indicate cells that lysed after mecillinam treatment in the PBP2^L61R culture. Scale bar equals 5 μm. d. Spot assay to test the sensitivities of PBP2^WT and PBP2^L61R to A22 (10 mg·L^-1) and mecillinam (2 mg·L^-1).