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In vivo properties of colicin A: channel activity and translocation across the envelope of
The influence of the dermonecrotic lethal toxin (approximately 120 kDa) produced by Pasteurella multocida serovarian D on planar phospholipid bilayers was studied. It was found that the toxin is able to increase the conductance of the bilayers by formation of low-conductive and cation-selective ion channels [27 pS at 4.0 M KCl, pH 7.5; zero current potential equals to -14.5 +/- 0.5 mV at threefold transmembrane gradient KCl (120 mM/40 mM)]. In biionic conditions the channels displayed weak selectivity between Na, K and Ca ions. The shapes of current-voltage characteristics (which were measured at different pH and salt concentrations) indicate that an energetic barrier for passing ions is situated near the center of the water pore of the ion channels. The effective diameter of the ion channel's water pore was established to be equal to 2.1 +/- 0.3 nm.
Colicins are unusual bacterial toxins because they are directed against close relatives of the producing strain. They kill their targets in one of three distinct ways; via a ribonuclease or deoxyribonuclease activity or by forming pores in the target cell's membrane. This review deals with the steps involved in pore-forming colicin activity including, initial synthesis of the toxin, toxin release, receptor binding, translocation across the periplasm and pore formation in the cytoplasmic membrane. Special reference is made to the role of colicin in vivo, the structural changes occurring during pore formation and the role of the immunity protein.
The kinetics of K+ efflux caused by colicin A in Escherichia coli-sensitive cells have been investigated by using a K(+)-selective electrode. The order of magnitude of the rate of K+ efflux per colicin molecule was comparable to that of ion channels. The dependence of K+ efflux upon multiplicity, pH, temperature, and membrane potential (delta psi) was determined. The translocation of colicin A from the outer membrane receptor to the inner membrane and insertion into the inner membrane required a fluid membrane, but once inserted, the channel properties showed little dependence upon the state of the lipids. At a given multiplicity, the lag time before the onset of K+ efflux was found to reflect the time required for translocation and/or insertion of colicin into the cytoplasmic membrane. Opening of the channel only occurred above a threshold value of delta psi of 85 +/- 10 and 110 +/- 5 mV at pH 6.8 and 7.8, respectively. Conditions were designed for closing and reopening of the channel in vivo. These conditions allowed us to test separately the delta psi requirements for translocation and channel opening: translocation and/or insertion did not appear to require delta psi. The channel formed in vivo featured properties similar to that of the channel in lipid planar bilayers.
The insertion of colicin A into monomolecular films and liposomes composed of different phospholipids was studied. Although colicin A was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged phospholipids such as phosphatidylglycerol. These interactions are highly dependent on the physical state of the lipid, the ionic strength, and the pH. Amino acid residues with a pK of 5.5 probably govern the lipid-protein interaction. At acidic pH, colicin A was able to insert into phospholipid vesicles and was as strong a penetrating agent as the lytic peptides bee venom mellitin and snake venom cardiotoxins. Below pH 5, colicin A induced aggregation and partial fusion of liposomes. At neutral and basic pH, colicin A penetration ability is limited, and the protein was unable to bind to phospholipid vesicles. However, association of colicin A with lipid vesicles could be achieved at pH 7 by the detergent dialysis technique. The apparent molecular area of colicin A inserted into phosphatidylglycerol films (2000 Å2/molecule) suggests that a substantial part of the colicin A molecule inserts into the lipid surface.
The study of colicin release from producing cells has revealed a novel mechanism of secretion. Instead of a built-in ‘tag’, such as a signal peptide containing information for secretion, the mechanism employs coordinate expression of a small protein which causes an increase in the envelope permeability, resulting in the release of the colicin as well as other proteins.On the other hand, the mechanism of entry of colicins into sensitive cells involves the same three stages of protein translocation that have been demonstrated for various cellular organelles. They first interact with receptors located at the surface of the outer membrane and are then transferred across the cell envelope in a process that requires energy and depends upon accessory proteins (TolA, TolB, TolC, TolQ, TolR) which might play a role similar to that of the secretory apparatus of eukaryotic and prokaryotic cells. At this point, the type of colicin described in this review interacts specifically with the inner membrane to form an ion channel.The pore-forming colicins are isolated as soluble proteins and yet insert spontaneously into lipid bilayers. The three-dimensional structures of some of these colicins should soon become available and site-directed mutagenesis studies have now provided a large number of modified polypeptides. Their use in model systems, particularly those in which the role of transmembrane potential can be tested for polypeptide insertion and ionic channel gating, constitutes a powerful handle with which to improve our understanding of the dynamics of protein insertion into and across membranes and the molecular basis of membrane excitability. In addition, their immunity proteins, which exist only in one state (membrane-inserted) will also contribute to such an understanding.
The bactericidal action of colicins K, E1, Ia, and other functionally related colicins involves disruption of active transport and leakage of ions from the cell. We show that a single colicin K molecule can form a voltage-dependent, relatively nonselective, ion-permeable channel of a few picosiemens conductance in a planar phospholipid bilayer membrane. In a membrane containing many of these channels, the ratio of the number of conducting to nonconducting channels changes e-fold per 3.7 mV. We suggest that the physiological effects of colicin K and functionally related colicins result from their ability to form ion-permeable channels in the bacterial plasma membrane.
Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin E1 by their respective immunity proteins.
TonB protein serves as an energy transducer to couple cytoplasmic membrane energy to high-affinity active transport of iron siderophores and vitamin B12 across the outer membranes of Gram-negative bacteria. The biochemical mechanism of the energy transduction remains to be determined, but important details are already known. TonB is targeted to and anchored in the cytoplasmic membrane by a single membrane-spanning domain and spans the periplasm to physically interact with outer-membrane receptors of the transport ligands. TonB-dependent energy transduction is modulated by ExbB protein, which stabilizes TonB, and possibly by several other proteins including ExbC, ExbD, and TolQ. TonB has a relatively short functional half-life that is accelerated when rates of active transport across the outer membrane are increased. A model that incorporates this information, as well as some tempered speculation, is presented.
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 74,683 D and that the immunity protein had a molecular weight of 10,057 D. 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.
A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor-binding properties of each protein were also analysed. From these results, we suggest that the NH2-terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor-binding domain. The COOH-terminal domain (residues 389 to 592) carries the pore-forming activity.