Energy-dependent Immunity Protein Release during tol-dependent Nuclease Colicin Translocation

Nuclease colicins bind their target receptor in the outer membrane of sensitive cells in the form of a high affinity complex with their cognate immunity proteins. Upon cell entry the immunity protein is lost from the complex by means that are poorly understood. We have developed a sensitive fluorescence assay that has enabled us to study the molecular requirements for immunity protein release. Nuclease colicins use members of the tol operon for their translocation across the outer membrane. We have demonstrated that the amino-terminal 80 residues of the colicin E9 molecule, which is the region that interacts with TolB, are essential for immunity protein release. Using tol deletion strains we analyzed the cellular components necessary for immunity protein release and found that in addition to a requirement for tolB, the tolA deletion strain was most affected. Complementation studies showed that the mutation H22A, within the transmembrane segment of TolA, abolishes immunity protein release. Investigation of the energy requirements demonstrated that the proton motive force of the cytoplasmic membrane is critical. Taken together these results demonstrate for the first time a clear energy requirement for the uptake of a nuclease colicin complex and suggest that energy transduced from the cytoplasmic membrane to the outer membrane by TolA could be the driving force for immunity protein release and concomitant translocation of the nuclease domain.Membrane translocation is a formidable challenge for folded proteins. Eukaryotes have an array of dedicated translocation machineries to accomplish this feat, for example during mitochondrial import of cytosolic precursor proteins for which it has recently become clear that there is a surprising diversity in targeting signals, import routes, and translocation complexes (1, 2). It is now widely accepted that the mitochondrial genome originated from within the (eu)bacterial domain of life, so it should perhaps not come as a surprise that certain features of mitochondrial import have evolved from these ancestors.Gram-negative bacteria possess two membranes to protect them from the external world, separated by a layer of peptidoglycan and the periplasmic space. Their outer membrane, with its asymmetrical composition of lipopolysaccharide (LPS)² and phospholipids, forms an impressive barrier to most substances with the exception of small hydrophilic nutrients that can diffuse through the resident porins (3). Processes that require an energy input at the outer membrane, such as iron siderophore uptake, therefore often rely on energy generated by ion gradients at the cytoplasmic membrane (4). Energy-transducing systems such as the ton and tol systems in Escherichia coli harvest energy generated at the cytoplasmic membrane and transduce it to the outer membrane. These two systems have a number of features in common, and cross-complementation between the two systems has been observed (5).The energy transducing capacity of the ton system is somewhat better defined and is accomplished by three proteins: the cytoplasmic membrane proteins ExbB and ExbD, which form a heteromultimeric complex that interacts with TonB (4). As a result, TonB undergoes a conformational change in response to the PMF of the cytoplasmic membrane, which allows it to traverse the periplasm and make contact with nutrient-loaded outer membrane receptors, thereby facilitating active import (6). The homology between ExbB/D, TolQ/R, and the PMF-responsive flagellar motor proteins MotA and MotB is well established, and the cumulative evidence now suggests that they act as energy-harvesting complexes (7–9). Evidence of an evolutionary relationship between TolA and TonB comes from work demonstrating structural similarities between the Pseudomonas aeruginosa TolAIII globular domain and the carboxyl-terminal domain of E. coli TonB despite the very low sequence conservation (10). The activities of TonB and TolA are also critically dependent on a conserved SHLS motif in their transmembrane region, the mutation of which affects the interaction with their respective energy-harvesting complexes (11, 12). The cellular function of the tol system in E. coli is, however, less clear. It is thought that the Tol proteins play a role in maintaining cell envelope integrity through a network of interactions spanning the cytoplasmic membrane, periplasm, and outer membrane (13).Both energy-transducing systems have been parasitized by the colicins, plasmid-encoded antibacterial proteins produced by E. coli, and phages for their translocation into the cell, but the energy requirements for these processes are not unequivocal (14). Group A colicins use the tol system and group B colicins the ton system in a process whereby interactions of their amino-terminal translocation domains with the Tol or Ton proteins in the periplasm ultimately lead to the entry of their carboxyl-terminal cytotoxic domain into the cell (15, 16). In common with most colicins, the DNase-type colicin E9 consists of three functional domains: the killing activity is contained in its carboxyl-terminal DNase domain; the central section contains the receptor-binding domain, which binds the vitamin B₁₂ receptor, BtuB, in the outer membrane; and the amino-terminal translocation domain is needed for the entry of the cytotoxic domain into the target cell. The first 83 residues of this translocation domain, commonly referred to as the NDR, contain the OmpF and TolB binding sites (17, 18). Upon synthesis colicin E9 forms a high affinity interaction with its cognate immunity protein, Im9, also encoded by the colicin operon. This heterodimeric complex formation protects colicin-producing cells against DNA damage and potential suicide prior to release of the complex in the environment. The nature of the complex formation between colicin E9 and Im9 and other colicin-immunity complexes has been well characterized, and in the case of colicin E9-Im9 the interaction is strong, as reflected by its dissociation constant on the order of 10⁻¹⁴ m under physiological conditions (19). Despite the high avidity of this interaction, the DNase domain of colicin E9 appears to have only a marginally stabilizing effect on Im9 (20).Currently much progress is being made to unravel the early events that take place after receptor binding, where it has been shown that the colicin E9 NDR enters the periplasm through the OmpF lumen where it interacts with TolB, possibly displacing it from its interaction with Pal (18, 21–24). It was also recently demonstrated that the receptor binding and translocation domains remain in contact with their binding partners in the outer membrane and the periplasm, respectively, when the DNase domain gains access to the cytoplasm (25). In contrast, the molecular mechanisms that govern the loss of the immunity protein from the colicin complex and the cell entry of the DNase domain are less well documented. Because of the strength of the interaction between the colicin and its cognate immunity protein, it has been proposed that removal of the immunity protein from the complex would require a cellular energy source. One recent report investigating immunity protein loss from the colicin E2-Im2 complex qualitatively concluded that receptor binding alone does not lead to immunity protein release and that a functional tol translocation complex is required to establish immunity protein release (26).Here we have presented data that for the first time demonstrate a role for the individual Tol proteins and address the issue of energy requirements for immunity protein release. We observed, by using a previously described disulfide-“locked” colicin construct and domain deletion mutants thereof, that entry of the amino-terminal 80 residues of the colicin translocation domain and its interaction with TolB are essential factors for immunity protein release. We have also demonstrated a crucial role for TolA and its transmembrane region in this process, showing that immunity protein release from the colicin complex is an energy-dependent process governed by the cytoplasmic membrane PMF. Finally we have provided a rationale for how an energized Tol system might lead to immunity protein loss and concomitant colicin uptake in sensitive cells.