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Once on the periplasmic side of the membrane, lipid II is polymerized by the glycosyltransferase (GT) and the transpeptidase (TP, the target of penicillin) activities of the penicillin-binding proteins (PBPs) 6, 7 in coordination with interacting proteins of their respective networks, allowing growth and division of the bacterial cell 2. The substrate for PG synthesis is the precursor lipid II, which is synthesized on the inner face of the cytoplasmic membrane and subsequently translocated through the membrane by a flippase (FtsW and/or MurJ) 3, 4, 5. These processes are performed by specialized multiprotein complexes, the elongasome and divisome each of them contains all the enzymatic activities required for the synthesis of new PG material and its insertion into the cell wall 2. In order to propagate, bacteria have to enlarge and divide their cell envelope including their PG sacculus 1. Most bacteria surround their cytoplasmic membrane with a peptidoglycan (PG) sacculus which protects the cell from bursting due to the turgor and maintains cell shape. This tight regulatory mechanism is consistent with the cell’s need to ensure appropriate use of the limited pool of lipid II. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. The divisome controls septal PG synthesis and separation of daughter cells. We suggest a two‐step model for bacterial division in which the Z‐ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles.Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. Consistently, a time delay of 14–21 min, depending on the growth rate, between Z‐ring formation and the mid cell recruitment of proteins down stream of FtsK was found. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno‐ and GFP‐fluorescence microscopy of steady state grown cells at a variety of growth rates. Summary Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. Nguyen‐Distèche, Martine den Blaauwen, Tanneke Piette, André Fraipont, Claudine Vinkenvleugel, Thessa M. Maturation of the Escherichia coli divisome occurs in two steps Maturation of the Escherichia coli divisome occurs in two stepsĪarsman, Mirjam E.