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

Lead Research Organisation: University of Oxford
Department Name: Biochemistry


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.

Technical Summary

(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


10 25 50
Description This grant aimed to explore aspects of the Gram-negative bacterial cell envelope and the outer membrane in particular, which is vital for their survival and makes them resistant to many types of antibiotics. Our approach was to use naturally occurring protein antibiotics (colicins) that circumnavigate across this membrane to illuminate properties of the outer membrane itself. The grant was very successful in terms of fundamental discovery that will have huge impact on this subject area. In addition to the many 'bread-and-butter' publications, we made two completely unexpected discoveries that have both been published in high impact journals:

1. We discovered how colicins thread an unstructured part of the protein through two pores of a three pore protein in the outer membrane. This allows the protein antibiotic a better foothold in the membrane and to capture a protein on the other side of the membrane in a defined orientation (published in Science)

2. Using fluorescently-labelled colicins in microscopy experiments we discovered that Gram-negative bacteria like E. coli organize their outer membrane proteins into large clusters, which has both a spatial and temporal dimension. One of the outcomes of such organization is that it helps bacteria change their outer membrane proteins quickly so helping them adapt to changes in the environment; for example, during treatment with antibiotics. This has completely revolutionised our understanding of the outer membrane itself, offers new routes for the creation of antibiotics to disrupt this organisation and poses many new questions (published in Nature).

3. Work from this award underpinned subsequent funding of a collaborative award from the Wellcome Trust (Protein Antibiotics)
Exploitation Route Our discoveries have raise many questions as to how and why bacteria organise their outer membrane proteins. We aim to address these questions in the coming years.

We are currently preparing further applications to explicitly investigate the effectiveness of bacteriocins as antibiotics to treat infections. Our work on transport may be used by biotech companies interested in delivering molecules into bacteria through outer membrane porins. We have obtained additional funding from bbsrc to study the underlying mechanism of this transport process, which was outside of the remit of the present application. We are also exploring whether some of our discoveries could be the subject of a patent.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description Advanced Grant
Amount € 2,000,000 (EUR)
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Description Goodger and Schorstein scholarship and EPA Cephalosporin Fund
Amount £24,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 12/2016
Description Molecular mechanisms of enterobacterial resistance to complement
Amount £950,416 (GBP)
Funding ID MR/R009937/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2018 
End 03/2021
Description Oxford lab move
Amount £150,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 03/2012 
End 08/2016
Description Protein Antibiotics: Discovery, mode of action and development
Amount £2,151,302 (GBP)
Funding ID 201505/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2016 
End 07/2022
Title Fluorescence labelling of bacterial outer membrane proteins 
Description Incorporating fluorophores into bacterial outer membrane proteins (OMPs) has proven problematic. GFP fusions do not generally work (unlike inner membrane proteins) because such fusions block the folding/maturation of the OMP. Covalent methods for labelling of specific OMPs are limited. We developed a method for non-covalent labelling of OMPs using colicins, supported by a BBSRC LoLa award (completed in 2015). We blocked the ability of colicins to import into bacteria using disulfide bonds while simultaneously incorporating organic fluorophore dyes into these molecules through cysteine labelling. Colicins bind with high affinity and specificity to particular OMPs, hence these reagents provided us with powerful fluorescence labels to follow the behaviour of these OMPs in live bacteria during cell growth and division. We have gone on to develop several such labels for many OMPs, in E. coli and P. aeruginosa. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact The development of fluorescently labelled colicins underpinned our discovery of binary OMP partitioning in E. coli. Through a combination of single molecule and ensemble fluorescence microscopy methods we demonstrated that the restricted diffusion of OMPs in this Gram-negative bacterium is due to the formation of large, supramolecular assemblies which we call OMP islands. We also discovered that OMP biosynthesis is biased towards the central regions of a dividing cell but absent at the cell poles. This results in binary partitioning of OMP islands during cell division. This mechanism is the basis for turnover of OMPs in the outer membrane of Gram-negative bacteria, which had never been explained previously. This work has implications for how Gram-negative bacteria adapt to a changing environment be it in an animal host or in the soil. 
Description Bioinformatics analysis of protein antibiotics 
Organisation University of Oxford
Department Department of Zoology
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a collaboration with Prof Martin Maiden. My laboratory brought the area of protein antibiotics to Prof Maiden's laboratory. I bring expertise in the protein chemistry and structure-function of protein antibiotics.
Collaborator Contribution Prof Maiden's laboratory contributed access to their BIGs database which includes the curated genomes of 1000s of bacterial genomes along with expertise in how to search this and other databases to find protein antibiotics (bacteriocins). This work has been supported by an EPSRC DTC studentship. Together we have identified many new bacteriocins and defined the bacterial phyla where such toxins are found.
Impact Manuscript being prepared currently
Start Year 2014
Description Electron microscopy structure analysis of ColE9 translocon complex 
Organisation Birkbeck, University of London
Department Department of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been collaborating with Prof Helen Saibil on the structure elucidation of the outer membrane translocon complex of ColE9 (~300 kDa). This followed the successful isolation of this multisubunit complex. My group have been testing purification regimes, detergents, amphipols etc to improve EM data.
Collaborator Contribution Dr Natasha Lukoyanova in Prof Saibil's lab has been testing our preparations of ColE9 translocon complex. She has recently collected cryo-EM data at Birkbeck which suggest we may be able to solve this structure by this technique. We already have high resolution 2D class averages and a low resolution electron density map. These data supported a successful application to Diamond to collect data on the Titan Cryos. Following this data collection we are currently developing a new purification strategy utilizing nanodiscs. These proved unsuccessful but we have had greater success with amphipols. Cryo-EM data have been collected and a structure is being refined.
Impact We have already published together on early negative EM images of the proteolysed ColE9 translocon (Housden et al (2013) Science)
Start Year 2013
Description Mathematical modelling 
Organisation Max Planck Society
Department Max Planck Institute for Terrestrial Microbiology
Country Germany 
Sector Academic/University 
PI Contribution Provision of outer membrane protein/lipoprotein fluorescence microscopy data from bacteria
Collaborator Contribution Dr Sean Murray has developed a novel mathematical procedure for extracting effective diffusion coefficients from FRAP data. this procedure helped us understand the functioning of the Tol-Pal system in Gram-negative bacteria.
Impact Szczepaniak et al (2020) Nature communications, In Press
Start Year 2019
Description Polymer supported membrane collaboration 
Organisation University of Osnabrück
Department School of Biology/Chemistry Osnabrück
Country Germany 
Sector Academic/University 
PI Contribution We collaborated with Prof Jacob Piehler's lab at the University of Osnabruck. My postdoc Patrice Rassam spent many weeks in Germany working with one of Prof Piehler's PhD student (Oliver Birkholz). They contributed substantially to the development of a polymer supported membrane system in which we reconstituted bacterial outer membrane proteins. This is highly specialised work, our work was significantly influenced by being able to work in the lab that developed this technology.
Collaborator Contribution Significant time on the part of the PhD student and significant resources given to the project (in particular single molecule imaging time, generation of supported membranes, lipids, and specifically labeled fluorescent proteins as controls).
Impact The main output is our Nature paper together
Start Year 2013
Description Simulations and mass spectrometry of outer membrane protein complexes 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution This collaboration relates to the laboratories of Prof Mark Sansom and Prof Carol Robinson in Oxford both of which work on membrane proteins and their associations. These collaborations began when my lab moved to Oxford in 2012 and continue to this date (2016). My laboratory contributed directly to both laboratories through the provision of materials and biological problems. In the case of Prof Robinson we have provided many outer membrane protein complexes that have contributed to the development of new mass spec methods for the analysis of membrane proteins (publication in press in Nat Methods 2016). In the case of Prof Sansom, my laboratory contributed data and ideas for the development of a new spatiotemporal model of the bacterial outer membrane in which outer membrane proteins (OMPs) are organised into large clusters. We contributed to the molecular dynamics simulations subsequently conducted by Prof Sansom's group. This work has been published in Nature in 2015.
Collaborator Contribution Native mass spec analysis of large membrane protein complexes (Robinson). Coarse-grained MD simulations of OMPs clusters (Sansom)
Impact Housden, N.G., Hopper, J.T.S., Lukoyanova, N., Rodriguez-Larrea, D., Wojdyla, J.A., Klein, A., Kaminska, R., Bayley, H., Saibil, H.R., Robinson, C.V. & Kleanthous, C. (2013) Intrinsically disordered protein threads through the bacterial outer membrane porin OmpF. Science 340, 1570-1574. This paper was selected as a 'Leading Edge' article on Intrinsically Disordered Proteins (2013) Cell 154, 473 Rassam, P., Copeland, N.A., Birkholz, O., Tóth, C., Chavent, M., Duncan, A.L., Cross, S.J., Housden, N.G., Kaminska, R., Seger, U., Quinn, D.M., Garrod, T.J., Sansom, M.S.P., Piehler, J., Baumann, C.G. & Kleanthous, C. (2015) Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria. Nature 523, 333-336. Subject of Trends in Microbiology Spotlight review by Rajeev Misra (2015) Entry and exit of outer membrane proteins. 23, 452 Gault J., Donlan, J.A.C., Liko,, I., Hopper, J.T.S., Gupta, K., Housden, N.G., Struwe, W., Marty, M.T., Mize, T., Bechara, C., Zhu, Y., Wu, B., Kleanthous, C., Damoc, E., Makarov, A. &. Robinson, C.V. (2016) High resolution mass spectrometry of lipids, peptides and drugs bound to membrane proteins. Nat Methods In Press
Start Year 2012
Description Terry and Masons Great Food Trip 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This engagement activity involved one of my DPhil students, Peter Holmes. Peter's PhD work is focused on protein-protein interactions of the Tol-Pal system in bacteria. Peter contributed to an episode (episode 15) of the Great British Food Trip, hosted by Terry Wogan and Mason McQueen, which sought to sample food from across the UK. Peter is a lover of baking and pizza making. The programme was aired in 2015 on BBC2 and hence had a national audience. The programme included an interview with Peter in my laboratory in the Department of Biochemistry where he explained his research interest on protein-protein interactions and how such interactions are important in the baking process, and then continued at Linacre college where Peter and his Society colleagues prepared pizzas.

Peter began the Linacre Pizza and Baking Society in 2014 (Linacre is his Oxford college) which has at its centre a scientific understanding of the baking process. They meet monthly to tackle different scientific concepts as they apply to Pizza. Foremost has been to teach an understanding of protein-protein interactions and how can we manipulate them to our advantage in dough making. Protein-protein interactions are the focus of research in my laboratory.

The advert for the Oxford segment of this series included a photograph of Peter with Sir Terry and Mason.
Year(s) Of Engagement Activity 2015