Most bacterial cells surround themselves with a peptidoglycan (PG) exoskeleton that confers them with shape and protection against lysis from internal turgor pressure. Class A Penicillin Binding Proteins (aPBPs) are a major family of PG synthases. Because they are targets of penicillin and related -lactam antibiotics, their biochemical and enzymatic functions are well known. However, their cellular function and regulation is still a major outstanding question. Here, we show that the cellular function of the aPBP, PBP1b, is the synthesis of the septal PG (sPG) wedge to reinforce the division site as visualized by in situ cryogenic electron tomography (cryo-ET) of Escherichia coli. Cells lacking PBP1b present weaker sPG and are prone to lysis due to cell envelope defects at the division site in vivo. We further show that such sPG reinforcement and building mechanism is independent of the lipofactor LpoB and PBP1a but dependent on the interaction between the intrinsically disorder domain (IDD) of the PBP1b-alpha isoform and the division protein FtsA for protein recruitment at the division site. This interaction is widely conserved among Enterobacteriaceae. Overall, our results support a model in which division of labor among PG synthetases is essential for the correct building of the sPG to effectively divide, and that the N-terminal IDD of PBP1b plays a major role in such molecular mechanism and division site recruitment coupled to Z-ring assembly.

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