Understanding Colicin N Import into Gram Negative Bacterial Cells using Small Angle Neutron Scattering

Abstract

Escherichia coli (E. coli) are gram-negative bacteria commonly found in human guts but which can cause food poisoning or severe systemic infections such as meningitis. Unlike other bacteria, gram-negative cells have an additional asymmetric outer membrane (OM) which consists of tightly-packed lipopolysaccharides in the outer leaflet of membrane and phospholipids on the inside. The OM is a selective permeability barrier and protects E. coli from antibiotics and bile acids. The occurrence of antibiotic-resistant bacteria is becoming increasingly serious and owing to the tightly-packed OM gram-negative bacteria can show even higher antibiotic resistance. In the search for novel effective antibiotics, Colicin N (ColN) is a promising model. ColN is a bacterial toxin produced and secreted by E. coli in time of stress. It translocates across the OM of target cells and kills via a voltage dependent channel in the inner membrane. ColN exploits outer membrane protein F (OmpF) as both a receptor and translocator. The pore-forming domain binds to the outside of OmpF (1, 2). Here we study the OmpF/ColN translocation complex in the presence of non-ionic detergents, where binding is driven by the translocation domain. The structure of complexes was determined by small angle neutron scattering using the SANS2D beam line and utilizing a contrast variation strategy with selective deuteration of proteins and Octyl Glucoside (OG). Mixing of three OG forms achieved exact contrast matches with the solvent. This enabled a low resolution structure of the translocon to be derived from ab initio modeling and revealed that the translocation and receptor-binding domains of ColN bind to the middle of the OmpF trimer.1. Baboolal, T. G. et al. Structure16, 371, (2008).2. Clifton, L. A. et al. J. Biol. Chem.287, 337,(2012).