Investigating E. coli cell envelope proteins and processes through colicin intoxication

Gram-negative bacteria have evolved to survive in diverse ecological niches. Many species are pathogenic while others are not, for example serving a symbiotic role in the mammalian gut helping to digest food. The major distinguishing feature of Gram-negative organisms compared to their Gram-positive counterparts is the existence of an additional membrane barrier, the outer membrane (OM), which is also responsible for the absence of staining with Gram dye in bacteriological procedures. Although serving an important barrier function for the organism, the OM is not an energised system. This presents significant problems for processes that require an energy source, such as the bringing in of essential nutrients that are too big to pass through the protein-pores that naturally exist in the OM. This is in contrast to the inner membrane (IM) of the bacterium which is an energised system by virtue of the organism's metabolism. An essential element of an energised IM is the flow of protons from the space between the OM and IM (the periplasm) back across the IM into the cell's cytoplasm, which is called the proton motive force (pmf). The pmf is a powersource for many energy-dependent processes in all organisms. In Gram-negative bacteria it is also responsible for the way in which the organism energises biochemical events at the OM, using long proteins that are embedded in the IM and which point towards the OM where they meet partner proteins. Two of the most important proteins that perform this type of energy linkage are TonB and TolA, each of which is part of larger protein assemblies usually referred to as the Ton and Tol systems. Ton is involved in bringing essential nutrients into the cell while Tol is involved in maintaining the barrier functions of the OM although how it does this is not clear. What is also not clear, even though this has been heavily studied for many years, is how these systems respond to pmf in a way that promotes their specific functions at the OM. This LOLA application aims to exploit the behaviour of a family of protein antibiotics called colicins to probe energy-dependent processes at the Gram-negative OM, focusing on Escherichia coli. Colicins are made by E. coli to kill neighbouring bacteria during times of competition and are very potent antibacterials; a single molecule entering the bacterium is sufficient to elicit cell death. Colicins begin their journey into an E. coli cell by binding to a nutrient receptor in the OM. Subsequent interactions with either the Ton or Tol systems catalyse their entry into the cell (a process called translocation) which is thought to be dependent on the pmf across the IM, but this has yet to be proven. We propose exploiting colicins as probes of OM processes using biochemical, biophysical and structural approaches. We will measure the forces that are exerted on colicins bound to the external surface of a cell and determine whether these forces are wholly pmf-dependent. We will establish how these potent antimicrobials use their associations with Tol proteins in the periplasm to penetrate the cells' OM defences, which may point the way toward new antibiotics. We will also capitalise on a remarkable series of observations that have for the first time visualised single colicin molecules bound to receptor proteins diffusing on the external surface of an E. coli cell. These observations highlight a property of the OM that contradicts standard biochemical and microbiological textbooks, where the motion of protein molecules embedded in the OM is assumed to be free and unrestricted, as is the case for the IM. In contrast, we find that movement is not unrestricted but rather demarcated into compartments. We will investigate the reason for such compartmentalisation and determine whether it plays a role in colicin translocation. Ultimately, this LOLA will provide fundamental new insight into the Gram-negative OM and its organisation.

(i)The textbook view of the E. coli OM is one where diffusion is unconstrained. Using single molecule TIRFM, we discovered highly restricted diffusion of the OM vitamin B12 receptor BtuB when bound to a fluorescently-labelled colicin. We will: -investigate the generality of restricted diffusion for OM receptors -make direct comparisons of OM and IM protein diffusion in the same cell -determine if there is correlated diffusion of OM and IM proteins during colicin translocation -test the hypothesis that constrained diffusion is due to OM interactions to the cell wall (ii)Using colicins as reporters of inside-out energy transduction in combination with uncouplers and mutants that disrupt Tol coupling to the pmf, we will address the relative contribution of active (pmf) versus passive forces to colicin translocation. We will: -use AFM to measure the forces in vitro across the non-covalent Tol complex and its complexes with colicins -use AFM to determine the kinetics of the colicin invasion pathway in vivo -use AFM to measure the forces experienced by a single colicin as it translocates across the OM -follow the unfolding of a single colicin molecule at the cell surface using TIRFM FRET (iii)We have discovered that the periplasmic protein TolB engages the IM protein TolA through a retractable N-terminal peptide analogous to the way in which OM nutrient receptors contact the IM protein TonB. We will: -determine the molecular basis of this recruitment for direct comparison to the Ton system -determine the structure of the TolAII-III protein -use newly devised assays to screen for endogenous ligands of the Tol system (iv)Colicins bind receptors in the OM and then subvert the Tol system for translocation. We will: -determine structures of colicins bound to OM proteins such as OmpF -solve the structure of the periplasmic trigger complex ColE9-TolB-TolA -use kinetic and thermodynamic analyses to understand how colicins manipulate Tol signalling