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.     

Daring to be different: colicin N finds another way

The mechanisms by which colicins, protein toxins produced by Escherichia coli, kill other E. coli, have become much better understood in recent years. Most colicins initially bind to an outer membrane protein receptor, and then search for a separate nearby outer membrane protein translocator that serves as a pathway into target cells. Many colicins use the outer membrane porin, OmpF, as that translocator, while using a different primary receptor. Colicin N is unique among known colicins in that only OmpF had been identified as being required for uptake of the colicin and it was presumed to somehow serve as both receptor and translocator. Genetic screens also identified a number of genes required for lipopolysaccharide (LPS) synthesis as uniquely required for killing by colicin N, but not by other colicins. Johnson et al. show that the receptor‐binding domain of colicin N binds to LPS, and does not require OmpF for that binding. LPS of a minimal length is required for binding, explaining the requirement for specific elements of the LPS biosynthetic pathway. For colicin N, the receptor‐binding domain does not recognize a protein, but rather the most abundant component of the outer membrane itself, LPS.     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Mechanisms of colicin binding and transport through outer membrane porins

To kill Escherichia coli, toxic proteins, called colicins, pass through the permeability barrier created by the outer membrane (OM) of the bacterial cell envelope. We consider a variety of different colicins, including A, B, D, E1, E3, Ia, M and N, that penetrate through the porins OmpF, FepA, BtuB, Cir and FhuA, to subsequently interact with a few targets in the periplasm, including TolA, TolB, TolC and TonB. We review the mechanisms, demonstrated and postulated, by which such toxins enter bacterial cells, from the initial binding stage on the cell surface to the internalization reaction through the OM bilayer. Our discussions endeavor to answer two main questions: what is the origin of colicin-binding affinity and specificity, and after adsorption to OM porins, do colicin polypeptides translocate through porin channels, or enter by another, currently unknown pathway?     

Assembly of colicin genes from a few DNA fragments. Nucleotide sequence of colicin D

The nucleotide sequence of a 2.4 kb Dral-EcoRV fragment of pColD-CA23 DNA was determined. The segment of DNA contained the colicin D structural gene (cda) and the colicin D immunity gene (cdi). From the nucleotide sequence it was deduced that colicin D had a molecular weight of 74683D and that the immunity protein had a molecular weight of 10057D. The amino-terminal portion of colicin D was found to be 96% homologous with the same region of colicin B. Both colicins share the same cell-surface receptor, FepA, and require the TonB protein for uptake. A putative TonB box pentapeptide sequence was identified in the amino terminus of the colicin D protein sequence. Since colicin D inhibits protein synthesis, it was unexpected that no homology was found between the carboxy-terminal part of this colicin and that of the protein synthesis inhibiting colicin E3 and cloacin DF13. This could indicate that colicin D does not function in the same manner as the latter two bacteriocins. The observed homology with colicin B supports the domain structure concept of colicin organization. The structural organization of the colicin operon is discussed. The extensive amino-terminal homology between colicins D and B, and the strong carboxy-terminal homology between colicins B, A, and N suggest an evolutionary assembly of colicin genes from a few DNA fragments which encode the functional domains responsible for colicin activity and uptake.     

Activity domains of the TonB protein

Escherichia coli and related Gram-negative bacteria contain an energy-coupied transport system through the outer membrane which consists of the proteins TonB, ExbB, ExbD anchored in the cytoplasmic membrane and receptors in the outer membrane. Differences in the activities of the Escherichia coli and the Serratia marcescens TonB proteins were used to identify TonB functional domains. In E. coli TonB segments were replaced by equivalent fragments of S. marcescens TonB and the activities of the resulting chimaeric proteins were determined. In addition, E. coli TonB was truncated at the C-terminal end, and point mutants were generated using bisulphite. From the results obtained we draw the following conclusions: an important site of interaction between TonB and ExbB is located in the M-terminal region of TonB within or close to the cytoplasmic membrane since an N-terminal 44-residue fragment of TonB was stabilized by ExbB and interfered with wild-type TonB activity. In addition, the activity of a TonB derivative in which histidine residue 20 was replaced by arginine was strongly reduced, and a double mutant containing arginine-7 to histidine and alanine-22 to threonine substitutions displayed an impaired uptake of ferrichrome. Furthermore, the domain around residue 160 is involved in TonB activity. S. marcescens TonB segments of this region in E. coli TonB conferred S. marcescens TonB activities, and E. coli TonB pöint mutants displayed strongly impaired activities for the uptake of colicin B and M and ferric siderophores. Plasmid-encoded tonB mutants of this region showed negative complementation of chromosomal wild-type tonB, and certain tonB mutants suppressed colicin B TonB-box mutants. Uptake of colicins required different domains in TonB, for colicin B and M around residue 160 and for colicin la, a domain closer to the C-terminal end. Tandem duplication of the E. coli (EP)X(KP) region by insertion of the S. marcescens (EP)×(KP) region (38 residues) and replacement of lysine residue 91 by glutamate did not alter TonB activity so that no evidence was obtained for this region to be implicated in receptor binding. The aberrant electrophoretic mobility of TonB was caused by the praline-rich sequence since its removal resulted in a normal mobility.     

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.     

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.     

Import of the transfer RNase colicin D requires site-specific interaction with the energy-transducing protein TonB

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous tonB mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both tonB mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.     

tRNA-targeting ribonucleases: molecular mechanisms and insights into their physiological roles

Most bacteria produce antibacterial proteins known as bacteriocins, which aid bacterial defence systems to provide a physiological advantage. To date, many kinds of bacteriocins have been characterized. Colicin has long been known as a plasmidborne bacteriocin that kills other Escherichia coli cells lacking the same plasmid. To defeat other cells, colicins exert specific activities such as ion-channel, DNase, and RNase activity. Colicin E5 and colicin D impair protein synthesis in sensitive E. coli cells; however, their physiological targets have not long been identified. This review describes our finding that colicins E5 and D are novel RNases targeting specific E. coli tRNAs and elucidates their enzymatic properties based on biochemical analyses and X-ray crystal structures. Moreover, tRNA cleavage mediates bacteriostasis, which depends on trans-translation. Based on these results and others, cell growth regulation depending on tRNA cleavage is also discussed.     

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.     

The crystal structure of the TolB box of colicin A in complex with TolB reveals important differences in the recruitment of the common TolB translocation portal used by group A colicins

Interaction of the TolB box of Group A colicins with the TolB protein in the periplasm of Escherichia coli cells promotes transport of the cytotoxic domain of the colicin across the cell envelope. The crystal structure of a complex between a 107‐residue peptide (TA_1–107) of the translocation domain of colicin A (ColA) and TolB identified the TolB box as a 12‐residue peptide that folded into a distorted hairpin within a central canyon of the β‐propeller domain of TolB. Comparison of this structure with that of the colicin E9 (ColE9) TolB box–TolB complex, together with site‐directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB. Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA. This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.     

Colicin Biology

Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.     

FtsH-dependent processing of RNase colicins D and E3 means that only the cytotoxic domains are imported into the cytoplasm

It has long been suggested that the import of nuclease colicins requires protein processing; however it had never been formally demonstrated. Here we show that two RNase colicins, E3 and D, which appropriate two different translocation machineries to cross the outer membrane (BtuB/Tol and FepA/TonB, respectively), undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms were identified in both colicin-sensitive cells and in cells immune to colicin because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm.     

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.     

Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system.

Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. Their mode of action involves three steps: binding to specific receptors located in the outer membrane, translocation across this membrane, and action on their targets. A specific colicin domain can be assigned to each of these steps. Colicins have been subdivided into two groups (A and B) depending on the proteins required for them to cross the external membrane. Plasmids were constructed which led to an overproduction of the Tol proteins involved in the import of group A colicins. In vitro binding of overexpressed Tol proteins to either Tol-dependent (group A) or TonB-dependent (group B) colicins was analyzed. The Tol dependent colicins A and E1 were able to interact with TolA but the TonB dependent colicin B was not. The C-terminal region of TolA, which is necessary for colicin uptake, was also found to be necessary for colicin A and E1 binding to occur. Furthermore, only the isolated N-terminal domain of colicin A, which is involved in the translocation step, was found to bind to TolA. These results demonstrate the existence of a correlation between the ability of group A colicins to translocate and their in vitro binding to TolA protein, suggesting that these interactions might be part of the colicin import process.     

The ecology and evolution of bacteriocins

In this review we focus on the ecological and evolutionary forces that determine the frequency and diversity of colicins inEscherichia coli. To begin, we describe that this killing phenotype is ubiquitous inE. coli, with as many as 50% of the isolates from a population producing colicin toxins, and that each population sampled has its own unique distribution of the more than 20 known colicin types. Next, we explore the dynamics of colicinogeny, which exhibits a typical form of frequency dependence, where the likelihood of successful colicin invasion into a population increases as the initial density of colicinogenic cells increases. We then incorporate thoughts on the evolution of chromosomal resistance to colicins and describe how resistance might influence the dynamics of colicinogen invasion and maintenance and the resulting colicin diversity. The final section deals with a genetic and phylogenetic characterization of colicins and a discussion of the evolutionary mechanisms responsible for generating colicin diversity. In this final section we provide details of the different molecular mechanisms known to play a role in generating colicin diversity, including the two most dominant forces in colincin evolution: recombination and positive, deversifying, selection.     

HIGH LEVELS OF COLICIN RESISTANCE IN ESCHERICHIA COLI

Colicins are plasmid-encoded antibiotics that are produced by and kill Escherichia coli and other related species. The frequency of colicinogeny is high, on average 30% of E. coli isolates produce colicins. Initial observations from one collection of 72 strains of E. coli (the ECOR collection) suggest that resistance to colicin killing is also ubiquitous, with over 70% of strains resistant to one or more colicins. To determine whether resistance is a common trait in E. coli, three additional strain collections were surveyed. In each of these collections levels of colicin production were high (from 15 to 50% of the strains produce colicins). Levels of colicin resistance were even higher, with most strains resistant to over 10 colicins. A survey of 137 non-E. coli isolates revealed even higher levels of resistance. We discuss a mechanism (pleiotropy) that could result in the co-occurrence of such high levels of colicin production and colicin resistance.     

Allosteric β‐propeller signalling in TolB and its manipulation by translocating colicins

The Tol system is a five‐protein assembly parasitized by colicins and bacteriophages that helps stabilize the Gram‐negative outer membrane (OM). We show that allosteric signalling through the six‐bladed β‐propeller protein TolB is central to Tol function in Escherichia coli and that this is subverted by colicins such as ColE9 to initiate their OM translocation. Protein–protein interactions with the TolB β‐propeller govern two conformational states that are adopted by the distal N‐terminal 12 residues of TolB that bind TolA in the inner membrane. ColE9 promotes disorder of this ‘TolA box’ and recruitment of TolA. In contrast to ColE9, binding of the OM lipoprotein Pal to the same site induces conformational changes that sequester the TolA box to the TolB surface in which it exhibits little or no TolA binding. Our data suggest that Pal is an OFF switch for the Tol assembly, whereas colicins promote an ON state even though mimicking Pal. Comparison of the TolB mechanism to that of vertebrate guanine nucleotide exchange factor RCC1 suggests that allosteric signalling may be more prevalent in β‐propeller proteins than currently realized.     

Dual roles of the central domain of colicin D tRNase in TonB-mediated import and in immunity

Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but it is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45-amino acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high affinity complex formation involves multiple interactions between these colicins and TonB. The central domain also contributes to the formation of the immunity complex, as well as being essential for uptake and thus killing. Colicin D is normally secreted in association with the immunity protein, and this complex involves the following two interactions: a major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the alpha4 helix of ImmD, which is on the opposite side of ImmD compared with the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity toward tRNAArg and a central domain, which can form a complex with ImmD but which does not function in TonB-mediated transport.