The DamX cell division protein of Escherichia coli: identification of amino acid residues critical for septal localization and peptidoglycan binding

In the bacterium Escherichia coli, cell division involves the concerted inward growth of all three layers of the cell envelope: the cytoplasmic membrane, the peptidoglycan (PG) cell wall, and the outer membrane. This is a complex, highly regulated process that involves over 20 proteins. Four of these proteins contain a domain of ~70 amino acids known as a SPOR domain (Pfam no. 05036). One of these SPOR domains (from a protein named FtsN) has been shown previously to bind PG. In this thesis we show that six additional SPOR domains, three from E. coli and three from other bacterial species, also bind PG. Thus, PG binding is a general activity of SPOR domains. We then examine the SPOR domain from DamX of E. coli in detail. In collaboration with Dr. Andrew Fowler of the NMR Core Facility, we determined the solution structure of the domain. The domain adopts an "RNP fold," characterized by a four-stranded anti-parallel β-sheet that is buttressed on one side by α-helixes. Several mutant forms of the DamX SPOR domain were constructed and studied both in vivo and in vitro. These studies support the following inferences: 1) The β-sheet is the PG-binding site; 2) The β-sheet contains critical information for targeting the SPOR domain to the midcell; 3) The SPOR domain probably localizes to the midcell by binding preferentially to septal PG; and 4) It follows, then, that septal PG must differ from PG elsewhere around the cell. We suggest that further studies of the SPOR:PG interaction will yield novel insights into PG biogenesis during septation.

This thesis also presents an in vivo characterization of several mutant forms of a cytoplasmic membrane protein named FtsW, homologs of which are found in all bacteria that contain a PG cell wall. FtsW recruits a PG synthase named FtsI to the division site and might also transport PG precursors across the cytoplasmic membrane. We systematically mutagenized each of FtsW's ten transmembrane (TM) helixes and investigated the ability of the mutant proteins to support division, localize to the division site, and recruit FtsI. This characterization leads us to propose that TM1 is involved in targeting FtsW to the division site, TM4 is involved in the putative transport activity, and TM10 is involved in recruitment of FtsI.