Divisome core complex in bacterial cell division revealed by cryo-EM

Cell division, or cytokinesis is a fundamental process of life and, in most bacteria, is driven by peptidoglycan synthesis at the septum1. It is catalysed by the divisome, a multi-protein complex with more than 20 components that spans the cell envelope in bacteria harbouring a cell wall2. Central to the divisome is the peptidoglycan-synthesising protein complex FtsWI, with the transglycosylase (TG) FtsW polymerising glycan strands from its substrate Lipid II3,4, and the transpeptidase (TP) FtsI crosslinking peptide stems, thus forming a covalent mesh between glycan strands5,6 (Fig. 1a). Septal peptidoglycan synthesis occurs after activation of the divisome glycosyltransferase-transpeptidase pair FtsWI3, in particular through an interaction with the heterotrimer FtsQBL7. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/517367v1_fig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1242fe6org.highwire.dtl.DTLVardef@c4d2c2org.highwire.dtl.DTLVardef@1e8eb0forg.highwire.dtl.DTLVardef@a02fff_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1:C_FLOATNO Biochemical and structural characterisation of the core divisome complex FtsWIQBL from P. aeruginosa. a) Septal peptidoglycan synthesis by FtsWIQBL during Gram-negative bacterial cell division. The transglycosylase FtsW (red), and transpeptidase FtsI (blue) bind the non-enzymatic subcomplex FtsQBL (green, violet and yellow, respectively). The complex contains 14 transmembrane helices - ten from FtsW and one each from FtsIQLB. The transglycosylase FtsW catalyses the polymerisation of GlcNAc-MurNAc disaccharides from Lipid II. The transpeptidase FtsI crosslinks the peptides from the nascent chain to adjacent peptides in the peptidoglycan layer between residues three and four. OM: outer membrane, IM: inner membrane, GlcNAc: N-acetylglucosamine, MurNAc: N-acetylmuramic acid, CC: coiled coil, TM: transmembrane. b) SDS-PAGE of the co-purified PaFtsWIQBL complex after size-exclusion chromatography. c) Western blot showing glycan strand ladders synthesised by divisome core complexes from Lipid II, demonstrating transglycosylase activity. The negative control does not contain any FtsWIQBL (lane 1). WT P. aeruginosa and E. coli FtsWIQBL complexes (lanes 2 and 4) are active transglycosylases, while the P. aeruginosa putative active site mutant FtsWD275AIQBL (lane 3) is inactive. d) Three representative 2D classes of our PaFtsWIQBL cryo-EM data. e) Left panel: side-view of the PaFtsWIQBL cryo-EM density at an overall resolution of 3.7 [A]. Protein colours are the same as those in a). Residual density from the detergent micelle is visible around the transmembrane domain in grey. Right panel: model of PaFtsWIQBL, rotated by 120{degrees} with respect to the density on the left-hand side. The putative FtsW active site residue D275 is indicated, as is the FtsI active site residue S294. The FtsW loop 219-233 and FtsI loop 45-50 are shown as a dotted line as they were too flexible to build. FtsQ and FtsQ were not resolved and are not shown. f) Top view of the periplasmic domain, showing interactions between FtsI, FtsL, FtsB and FtsQ. C_FIG Here, we present the cryo-EM structure of the catalytic divisome core complex FtsWIQBL from Pseudomonas aeruginosa at 3.7 [A] resolution. The structure reveals the intricate details of the periplasmic interfaces within FtsWIQBL, including the positioning of FtsI by the coiled coil of FtsBL, as well as a transmembrane domain containing FtsWIBL but not FtsQ. With our structure we are able to provide molecular mechanisms of a multitude of known mutations that interfere with divisome activation and regulation. Finally, we reveal a large conformational switch between presumably inactive and active states of the FtsWI core enzymes. Our work is foundational for further structural, biochemical and genetic studies elucidating the molecular mechanisms of bacterial cell division. Since the divisome peptidoglycan synthase is essential for cell division in most bacteria, and is absent in eukaryotic cells entirely, it is a key target of important antibiotics and antibiotic development8, and we suggest that our structure will help to accelerate these efforts.