Direct interaction between MreB and the RodA‐PBP2 complex organizes lateral cell wall synthesis in <i>Escherichia coli</i>

Abstract The elongasome, or the Rod complex, orchestrates lateral peptidoglycan (PG) synthesis in many rod‐shaped bacteria. It consists of the actin‐like protein MreB, the PG synthase RodA‐PBP2 complex, as well as MreCD and RodZ. Although the loss or disruption of any elongasome component results in a loss of rod shape, previous studies found that a constitutively active RodA‐PBP2 complex can partially bypass the requirement of MreCD and RodZ for lateral PG synthesis and restore rod shape. However, how MreB is connected to RodA‐PBP2 under this situation and whether this linkage is important for elongasome activity in wild‐type cells remain unknown. Here, we report the isolation of additional RodA and PBP2 variants that can partially compensate for the absence of MreCD and RodZ in lateral PG synthesis. Taking advantage of these mutants and guided by an AlphaFold 3 structural model of the elongasome complex, we discover that both the cytoplasmic region of PBP2 and the C‐terminal tail of RodA interact with MreB. Moreover, disruption of these interactions results in a loss of rod shape, indicating that the interaction between MreB and RodA‐PBP2 is critical for elongasome function. Taken together, our results uncover how the MreB cytoskeleton is coupled to RodA‐PBP2 to organize lateral PG synthesis. These findings provide mechanistic insights into cell wall biogenesis in bacteria and offer strategies for the development of new antibiotics targeting the elongasome.