The peptidoglycan cell wall is essential for the survival and morphogenesis of bacteria.1 For decades it was thought that only class A penicillin-binding proteins (aPBPs) and related enzymes effected peptidoglycan synthesis. Recently, it was shown that RodA, a member of the unrelated SEDS protein family, also acts as a peptidoglycan polymerase.2–4 Not all bacteria require RodA for growth; however, its homologue, FtsW, is a core member of the divisome complex that appears to be universally essential for septal cell wall assembly.5,6 FtsW was previously proposed to translocate the peptidoglycan precursor Lipid II across the cytoplasmic membrane.7,8 We report here that purified FtsW polymerizes Lipid II into peptidoglycan, but show that its polymerase activity requires complex formation with its partner class B PBP (bPBP). We further demonstrate that the polymerase activity of FtsW is required for its function in vivo. Thus, our findings establish FtsW as a peptidoglycan polymerase that works with its cognate bPBP to produce septal peptidoglycan during cell division.

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery.

The S hape, E longation, D ivision, and S porulation (“SEDS”) proteins are a highly conserved family of transmembrane glycosyltransferases that work in concert with class B penicillin binding proteins (bPBPs) to build the bacterial peptidoglycan cell wall 1 – 6 . How these proteins coordinate polymerization of new glycan strands with their crosslinking to the existing peptidoglycan meshwork remains unclear. Here, we report the crystal structure of the prototypical SEDS protein RodA from Thermus thermophilus in complex with its cognate bPBP at 3.3 Å resolution. The structure reveals a 1:1 stoichiometric complex with two extensive interaction interfaces between the proteins: one in the membrane plane and the other at the extracytoplasmic surface. When in complex with a bPBP, RodA shows a ~10 Å shift of transmembrane helix 7 that exposes a large membrane-accessible cavity. Negative-stain electron microscopy reveals that the complex can adopt a variety of different conformations. These data define the bPBP pedestal domain as the key allosteric activator of RodA both in vitro and in vivo , explaining how a SEDS:bPBP complex can coordinate its dual enzymatic activities of peptidoglycan polymerization and crosslinking to build the cell wall.