Protein translocation across the gram negative bacterial membrane /

Abstract

Gram-negative bacteria are becoming increasingly resistant to many antibiotics. Unlike other bacteria, gram-negative bacteria have an additional membrane, the so-called outer membrane, which protects them from harmful agents. This membrane is highly asymmetric and contains tightly-packed lipopolysaccharides (LPS) in the outer leaflet and phospholipids in the inner leaflet. This thesis describes a study of the outer membrane protein F (OmpF) which is the most abundant porin present in the outer membrane of Esherichia coli. OmpF plays a key role in the organization of outer membrane and is also a receptor and translocator for colicins, which are antibacterial toxins. The aim of this project is to better understand the defensive barrier of gram-negative bacteria and find routes to pass through it by studying the interaction of OmpF with colicin N (ColN) and LPS. The solution structure of ColN and the OmpFColN complex in neutral detergents, studied by AUC and small-angle scattering (SAS), indicated that translocation and receptor binding domain of ColN (ColNTR) became more compact when binding to outside of OmpF. Furthermore, OmpF in complex with the protein TolA, which forms part of the ColN translocon complex, was also studied by SAS. It showed that TolA bound to OmpF also became more compact though TolA remains a flexible structure. Furthermore, the interaction of OmpF with LPS was studied. Mutagenesis of positivelycharged residues on OmpF was used to disrupt its electrostatic interaction with LPS. The findings suggested that OmpF has two LPS-binding sites and that OmpF binds to LPS via the minimal Lipid A moiety. This was supported by SAS data. Dynamic light scattering experiments indicated that OmpF, LPS and divalent cations form larger-scale structures, which are reminiscent of the outer membrane of bacteria. Alternative approaches for studying OmpF in amphipol (APol) and nanodisc were also utilised. OmpF/APol assembled as filaments in the absence of free APol but these converted into 2D arrays in the presence of LPS and calcium ions. OmpF could be incorporated into Nanodiscs, significantly increasing their diameters compared to empty Nanodiscs.