Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF.

Marie‐Louise R Francis,Nicholas G Housden,Emma Elliston,Boonyaporn Chinthammit,Natalya Lukoyanova,Renata Kaminska,Melissa N Webby,Colin Kleanthous

doi:10.15252/embj.2021108610

Marie‐Louise R Francis, Nicholas G Housden + Show 6 more

Open Access

https://doi.org/10.15252/embj.2021108610

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Abstract

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo‐EM and by imaging toxin import, we uncover the mechanism by which the Tol‐dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9’s disordered N‐terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton‐motive force, which is delivered by the TolQ‐TolR‐TolA‐TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol‐ and Ton‐dependent bacteriocins cross the bacterial outer membrane.

Highlights

The asymmetric outer membrane (OM) of Gram-negative bacteria, composed of lipopolysaccharides (LPS) in the outer leaflet and phospholipids in the inner leaflet, shields the organism against environmental insults, the immune systems of plants and animals, bile salts in the human gut and several classes of antibiotics (Nikaido, 2003; Whitfield & Trent, 2014; Ranf, 2016; Vergalli et al, 2020)
Immunity proteins are high-affinity inhibitors that are co-expressed with the colicin and protect the producing cell from its cytotoxic action (Kleanthous et al, 1999), but are jettisoned during import of the bacteriocin (Vankemmelbeke et al, 2013)
At its N-terminus is an 83 amino acid intrinsically unstructured translocation domain (IUTD) that houses three protein-protein interaction epitopes (Housden et al, 2010); a 16-residue TolB-binding epitope (TBE) associates with the periplasmic protein TolB which is flanked by two OmpF-binding sites (OBS1 and OBS2)

Summary

Introduction

The asymmetric outer membrane (OM) of Gram-negative bacteria, composed of lipopolysaccharides (LPS) in the outer leaflet and phospholipids in the inner leaflet, shields the organism against environmental insults, the immune systems of plants and animals, bile salts in the human gut and several classes of antibiotics (Nikaido, 2003; Whitfield & Trent, 2014; Ranf, 2016; Vergalli et al, 2020). Attack strategies are typically of two types, those that depend on physical or close contact between bacterial cells and those mediated by diffusible molecules (Ruhe et al, 2020). The former includes contact-dependent inhibitors, where a toxin projected from an attacking cell binds a surface receptor of a recipient cell prior to import (Aoki et al, 2010), and type VI secretion, where toxins are delivered by a needle that punctures the OM (Basler et al, 2013). We focus on a family of protein bacteriocins from the antimicrobial armoury of the Enterobacteriaceae and reveal their convoluted use of porins for OM transport

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