PBP3 interacts in vitro with FtsW 12, PBP1b 15, and FtsN 16 and with other proteins of the divisome 2. PBP1b requires the PG TPase PBP3 which itself requires FtsW for septal localization 14, 15. The bifunctional PG GTase/TPase PBP1b and its regulators (LpoB, CpoB, TolA) also associate with the divisome. Later, the downstream components FtsK, FtsQ-FtsL-FtsB, FtsW-FtsI (PBP3) and FtsN join sequentially, some as preformed subcomplexes 11, 12, to form the mature divisome 13. In Escherichia coli, the divisome includes over 20 proteins which assemble in an ordered and interdependent manner in two steps 10 first, the tubulin-like FtsZ, ZipA, FtsA, ZapA-E and FtsEX localize at mid-cell underneath the cytoplasmic membrane. This organization allows the coordination between the cytoskeleton and the synthesis of precursors in the cytoplasm, their transport across the cytoplasmic membrane and septal peptidoglycan synthesis, and, in Gram-negative bacteria, the coordination with outer membrane invagination 8, 9. The components of the divisome span the cytoplasm, the cytoplasmic membrane, the periplasm and the outer membrane (in Gram-negative bacteria) and communicate by dynamic protein-protein interactions through these cell compartments. The divisome controls septal PG synthesis at mid-cell and its differentiation into the new poles of daughter cells. 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. Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division.