MreC and MreD balance the interaction between the elongasome proteins PBP2 and RodA

Author summary The cell envelope of Escherichia coli bears the protective and shape-determining peptidoglycan layer sandwiched between the outer and inner membranes. Length growth in bacteria is accomplished by a protein complex termed elongasome. In this complex, RodA and PBP2 provide the peptidoglycan glycosyltransferase and transpeptidase activities needed to synthesize new peptidoglycan during length growth, respectively, and PBP2 activates RodA. Using Forster Resonance Energy Transfer (FRET) that reports not only on whether proteins interact with each other but also on conformational changes during interactions, we investigated how RodA and PBP2 interact. We show that the interactions between MreC and MreD with PBP2-RodA alter the nature of the interaction between PBP2 and RodA and hypothesize that the corresponding conformational change in the PBP2-RodA complex allows switching between the 'on' and 'off' states of PBP2 activity, contributing to the regulation of elongasome activity. Rod-shape of most bacteria is maintained by the elongasome, which mediates the synthesis and insertion of peptidoglycan into the cylindrical part of the cell wall. The elongasome contains several essential proteins, such as RodA, PBP2, and the MreBCD proteins, but how its activities are regulated remains poorly understood. Using E. coli as a model system, we investigated the interactions between core elongasome proteins in vivo. Our results show that PBP2 and RodA form a complex mediated by their transmembrane and periplasmic parts and independent of their catalytic activity. MreC and MreD also interact directly with PBP2. MreC elicits a change in the interaction between PBP2 and RodA, which is suppressed by MreD. The cytoplasmic domain of PBP2 is required for this suppression. We hypothesize that the in vivo measured PBP2-RodA interaction change induced by MreC corresponds to the conformational change in PBP2 as observed in the MreC-PBP2 crystal structure, which was suggested to be the "on state" of PBP2. Our results indicate that the balance between MreC and MreD determines the activity of PBP2, which could open new strategies for antibiotic drug development.