Molecular mechanism of bacteriocin import into bacterial cells. (360G-Wellcome-082045_A_07_Z)

Our proposal brings together expertise in biochemistry/structural biology (York), microbiology (Nottingham) and spectroscopy (Norwich) to determine how nuclease colicins penetrate the cell defences of bacteria. The programme has four key goals: (1) To establish the mechanism by which nuclease colicins translocate acrossthe E. coli outer membrane, centred on a structural dissection of the multiprotein, membrane-bound complex that assembles prior to import. (2) To determine the function of the periplasmic Tol-Pal complex in conjunction with how and why it is parasitised by translocating colicins, using microscopy to determine whether colicins target the bacterial septation machinery, biochemical analysis of the overexpressed membrane-bound TolQRA complex, along with functional dissection of TolB through spectroscopic and biophysical analysis. (3) To establish the mechanism by which colicin nucleases retro-translocate across the bacterial inner membrane using a range of mutants, cell-based assays and crosslinking that will probe the role of electrostatics and the AAA+ATPase FtsH in membrane trafficking. (4) To use NMR to define the conformational ensemble of the natively disordered N-terminal domains of Ton- and Tol-dependent colicins that recruit protein partners for import and examine TolB binding mechanism using pre-steady state methods.