The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein

Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low‐copy‐number, high‐affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high‐affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor‐binding domain (ColN‐R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid‐ and solid‐state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPS^ΔLIPID) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.     

Colicin N binds to the periphery of its receptor and translocator, outer membrane protein F

Colicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusually simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. Studies of this binary complex may therefore reveal a significant component of the translocation pathway. Here we show that, in 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface. The major lipid of the outer leaflet interface is lipopolysaccharide (LPS). It is further shown that colicin N binding displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. Colicin N also binds artificial OmpF dimers, indicating that trimeric symmetry plays no part in the interaction. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport.     

Colicin import and pore formation: a system for studying protein transport across membranes?

Pore-forming colicins are a family of protein toxins (M_r40–70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage-gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient-uptake systems and the so-called‘Tol’proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage-gated channels). The Tol-dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α-helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin-resistant mutant of OmpF. The X-ray crystal structure of the channel-forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site-directed muta-genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N-terminal domains of colicins interact with the lumen of the OmpF pore and then with the C-terminal domain of TolA. A molten globular conformation of colicins probably constitutes the intermediate translocation/insertion competent state. Once the pore has formed, the polypeptide chain spans the whole cell envelope. Three distinct steps occur in the last stage of the process: (i) fast binding of the C-terminal domain to the outer face of the cytoplasmic membrane; (ii) a slow insertion of the polypeptide chain into the outer face of the inner membrane in the absence of Δψ and (iii) a profound reorganization of the helix association, triggered by the transmembrane potential and resulting in the formation of the colicin channel.     

Comparison of the uptake systems for the entry of various BtuB group colicins into Escherichia coli

Colicins A, E1, E2 and E3 belong to the BtuB group of colicins. The NH2-terminal region of colicin A is required for translocation, and defects in this region cannot be overcome by osmotic shock of sensitive cells. In addition to BtuB, colicin A requires OmpF for efficient uptake by sensitive cells. The roles of BtuB and OmpF in translocation and binding to the receptor of the colicins A, E1, E2 and E3 were compared. The results suggest that for colicin A OmpF is used both as a receptor and for translocation across the outer membrane. In contrast, for colicin E1, OmpF is used neither as a receptor nor for translocation. For colicins E2 and E3, the situation is intermediate: only BtuB is used as a receptor but both BtuB and OmpF are involved in the translocation step.     

The colicin Ia receptor,Cir, is also the translocator for colicin Ia

Colicin Ia, a channel‐forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill Escherichia coli, it must cross this membrane. The crystal structure of Ia receptor‐binding domain bound to Cir, a 22‐stranded plugged β‐barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no ‘second receptor’ has ever been identified for TonB‐dependent colicins, such as Ia. We show that if the receptor‐binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild‐type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N‐terminal translocation (T) domain and its channel‐forming C‐terminal domain. Deletion of colicin Ia's receptor‐binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia's receptor‐binding domain, Cir also binds weakly to its translocation domain.     

Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import

The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.     

Translocator hunt comes full Cir‐Col

The ability of Escherichia coli to kill other E. coli using protein antibiotics known as colicins has been known for many years, but the mechanisms involved poorly understood. Recent progress has been rapid, however, particularly concerning events on either side of the outer membrane (OM). Structures of colicins bound to OM receptors have been determined and we have detailed mechanistic information on how colicins subvert the periplasmic complexes of TolQRAB/Pal or TonB/ExbB/ExbD to trigger cell entry. In this issue of Molecular Microbiology, Jakes and Finkelstein answer a long‐standing problem concerning the uptake mechanism of the pore‐forming colicin ColIa: How does the TonB box of the colicin cross the OM following high‐affinity binding of ColIa to its primary receptor, the siderophore transporter Cir? Through a series of chimeric protein constructions tested for their activity against a range of mutants and in cell death protection assays, the authors come up with the surprising observation that following binding of ColIa to Cir it recruits another Cir protein as its OM translocator. Not only does this settle various conundrums in the literature, but the translocation mechanism that stems from their study will likely be applicable to many TonB‐dependent colicins.     

Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K12

Summary To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2–73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, (iii) that colicin N uses OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.     

Mobility of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex

Diffusion of two Escherichia coli outer membrane proteins—the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages—was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient (D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05 ± 0.01 μm²/s and 0.10 ± 0.02 μm²/s, respectively, over a timescale of 25-150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27 ± 0.06 μm²/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006 ± 0.002 μm²/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.     

The unstructured domain of colicin N kills Escherichia coli

Bacteria often produce toxins which kill competing bacteria. Colicins, produced by and toxic to Escherichia coli bacteria are three‐domain proteins so efficient that one molecule can kill a cell. The C‐terminal domain carries the lethal activity and the central domain is required for surface receptor binding. The N‐terminal domain, required for translocation across the outer membrane, is always intrinsically unstructured. It has always been assumed therefore that the C‐terminal cytotoxic domain is required for the bactericidal activity. Here we report the unexpected finding that in isolation, the 90‐residue unstructured N‐terminal domain of colicin N is cytotoxic. Furthermore it causes ion leakage from cells but, unlike known antimicrobial peptides (AMPs) with this property, shows no membrane binding behaviour. Finally, its activity remains strictly dependent upon the same receptor proteins (OmpF and TolA) used by full‐length colicin N. This mechanism of rapid membrane disruption, via receptor mediated binding of a soluble peptide, may reveal a new target for the development of highly specific antibacterials.     

Colicin E2 is still in contact with its receptor and import machinery when its nuclease domain enters the cytoplasm

Colicins reach their targets in susceptible Escherichia coli strains through two envelope protein systems: the Tol system is used by group A colicins and the TonB system by group B colicins. Colicin E2 (ColE2) is a cytotoxic protein that recognizes the outer membrane receptor BtuB. After gaining access to the cytoplasmic membrane of sensitive Escherichia coli cells, ColE2 enters the cytoplasm to cleave DNA. After binding to BtuB, ColE2 interacts with the Tol system to reach its target. However, it is not known if the entire colicin or only the nuclease domain of ColE2 enters the cell. Here I show that preincubation of ColE2 with Escherichia coli cells prevents binding and translocation of pore-forming colicins of group A but not of group B. This inhibition persisted even when cells were incubated with ColE2 for 30 min before the addition of pore-forming colicins, indicating that ColE2 releases neither its receptor nor its translocation machinery when its nuclease domain enters the cells. These competition experiments enabled me to estimate the time required for ColE2 binding to its receptor and translocation.     

Increased production of the outer membrane receptors for colicins B, D and M by Escherichia coli under iron starvation.

Growth of $\text{E. coli}$ K-12 under severe iron stress results in increased production of the outer membrane receptors for colicins B, D, Ib and M. The increase in colicin receptor activity coincides with the appearance of large amounts of two high molecular weight proteins in the outer membrane of the cells. These proteins are identified as the outer membrane receptors for colicins B and D and for colicin M. Mutants lacking a functional outer membrane receptor for colicins B and D are defective in the uptake of iron complexed with the siderochrome enterochelin, and are thus comparable with $\text{tonA}$ mutants which lack a functional receptor for colicin M and are defective in the uptake of iron complexed with ferrichrome (6). The colicin B and D receptor may therefore function in the uptake of ferri-enterochelin.     

Bdellovibrio possesses a prey-derived OmpF protein in its outer membrane

Bdellovibrio bacteriovorus 109D andBdellovibrio stolpii derive one of their major outer membrane proteins from the outer membrane of their prey. This prey-derived protein corresponds to the OmpF protein ofEscherichia coli. Bdellovibrios cultivated onSalmonella typhimurium prey acquire theSalmonella OmpF protein; this protein is distinguishable electrophoretically from the OmpF protein ofE. coli. Bdellovibrios containing the prey-derived OmpF protein are sensitive to killing by colicin A but not colicin E1, whereas bdellovibrios without this protein are completely resistant to colicin killing.     

Crystal structures of the OmpF porin: function in a colicin translocon

The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 A OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co-crystallization of OmpF with the unfolded 83 residue glycine-rich N-terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 A structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly-glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno-extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.     

Binding domains of colicins E1, E2 and E3 in the receptor protein btub ofEscherichia coli

Eight reagents specifically modifying amino acids were applied to cells of a standardEscherichia coli colicin indicator strain to followin vivo changes of its binding capacity for colicins E1–E3 and hence the binding domains (epitopes) for them in the outer membrane receptor protein BtuB. The effect of these reagents was also investigated in a mutant strain carrying an extensive BtuB deletion. The following differences of the binding epitopes could be ascertained.Colicin E1: Blockage of OH-groups, just as N-substitution of His and modification of Arg and Trp enhance binding of colicin E1. In the deleted receptor, also abolition of carboxylic anion bonds enhances its affinity for colicin E1. It follows that colicin E1 is bound, most of all, to the hydrophobic domain A (loops 1+2) of BtuB.Colicins E2 and E3: both exert rather analogous binding parameters. In contrast to E1, O-substitution of Ser and Thr dramatically decreases the E2 and E3 binding, similarly to modification of Lys. There is also a clear difference in the binding affinity of the domain for E2 and/or E3 and for E1 following modifications of their Arg and His. Colicins E2 and E3 are rather bound to the hydrophilic domain B (loops 5–7) of the receptor. In this respect, interactions of colicins E2 and E3 with deeper parts of A and B domains (Trp, several Arg, Lys and His residues) exhibited subtle differences. Acidic pH (4.5–6.0) shows a positive, while pH 7.0–8.5 a rather negative impact on the receptor-binding function for the colicins. It was clearly demonstrated that there is just a partial difference between the binding behavior of colicins E1, E2 and/or E3.     

Involvement of OmpF during reception and translocation steps of colicin N entry

[125I]-colicin N binds to OmpF receptor sites (70,000 per cell) with an average Kassoc of 3.2 x 10(6) M-1 at 23 degrees C. Monoclonal antibody directed against a cell-surface-exposed epitope of OmpF is able to complete with the binding of the colicin in vitro and also to protect against colicin N in vivo. OmpF is an absolute requirement for colicin N uptake. OmpC cannot serve as a substitute for OmpF during translocation across the outer membrane under receptor bypass conditions, which is in contrast to colicin A. Colicin N does not cross-react with various monoclonal antibodies directed against colicin A.     

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

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.     

Intracellular Immunization of Prokaryotic Cells against a Bacteriotoxin

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E. coli. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.     

Colicin Ia and Ib binding to Escherichia coli envelopes and partially purified cell walls

The specific binding of ¹²⁵ Iodine labelled colicin Ia and Ib to Escherichia coli cell envelopes and partially purified cell walls is demonstrated. Neither partially purified cytoplasmic membranes isolated from a wild type sensitive strain nor envelopes or cell walls prepared from an E. coli mutant known to be defective in the colicin I receptor could bind the colicins. Competition studies suggest that colicins Ia and Ib have a common bacterial receptor which resides in the bacterial cell wall.