Site-directed Mutagenesis of the SPOR Domain from Escherichia coli FtsN

Cell division in Escherichia coli involves more than 30 proteins that localize to the midcell (septum) to form the septal ring. Most of these proteins bind other division proteins to localize to the midcell, but proteins that contain a “SPOR domain” may possibly localize by a completely different mechanism. These SPOR domains are thought to bind to septal peptidoglycan.  Over 500 bacterial species, including many pathogens, are known to have proteins with a SPOR domain, which argues that they are important. The best studied of the E. coli SPOR domain proteins is named FtsN, which has a SPOR domain at its C-terminus (Ursinus et al., 2004). The structure of the SPOR domain from FtsN has been determined by Nuclear Magnetic Resonance (NMR) and comprises a 4-stranded antiparallel β-sheet flanked on one side by two α-helices (Yang et al., 2005).  We hypothesize that the SPOR domain from FtsN recognizes and binds septal PG. To gain some insight into how SPOR domains localize to septal peptidoglycan, we used degenerate primers to mutagenize 33 amino acids with surface-exposed R-groups, thereby creating a set of 92 mutant FtsN SPOR domains.  The domains were fused to Tat-targeted GFP and assayed for septal localization in live cells.  Western blotting with anti-GFP antibody was used to assess proper production of the mutant proteins.  These studies revealed 4 amino acids that are specifically required for efficient septal localization.  All of these critical residues are in the β-sheet, which we propose is the peptidoglycan binding site.