Adenosine 5''-triphosphate-linked transhydrogenase in cytoplasmic membranes of colicin-treated and untreated Escherichia coli.

The adenosine 5'-triphosphate (ATP)-linked transhydrogenase reaction, present in the particulate fractions of Escherichia coli, was previously shown to be inhibited in these fractions when the bacteria were treated with colicins K or El. The purpose of this study was to characterized the ATP-linked transhydrogenase reaction and the colicin-caused inhibition of the reaction in purified cytoplasmic membranes. Particulate fractions from bacteria treated or untreated with colicins were separated on sucrose gradients into cell wall membrane and cytoplasmic membrane fractions. The ATP-linked transhydrogenase reaction was found to be exclusively associated with the cytoplasmic membrane fractions. The reaction was inhibited by carbonylcyanide m-chlorophenlhdrazone, dinitrophenol, N,N'-dicyclohexylcarbodiimide, and trypsin. Although the cytoplasmic membrane fractions were purified from the majoriy of the cell wall membrane and its bound colicins, they showed the inhibitory effects of colicins K and El on the ATP-linked transhydrogenase reaction. The inhibition of ATP-linked transhydrogenase reaction induced by the colicin could not be reversed by subjection the isolated membranes to a variety of physical and chemical treatments. Cytoplasmic membranes depleted of energy-transducing adenosine triphosphatase ATPase) complex (coupling factor) lost the ATP-linked transhydrogenase activity. The ATPase complexes isolated from membranes of bacteria treated or untreated with colicins El or K reconstituted high levels of ATP-linded transhydrogenase activity to depleted membranes of untreated bacteria. The same ATPase complexes reconstituted low levels of activity to depleted membranes of the treated bacteria.     

Colicins and bacterial membranes: structures and functions. II. Studies on reconstituted homologous and hybrid membranes prepared from cytoplasmic membranes of untreated and colicin K-treated bacteria.

Cytoplasmic membranes of Escherichia coli K12 C600 treated and not treated with colicin K were dissociated into unsolubilized and solubilized fractions. Neither fraction catalyzed ATP-linked transhydrogenase activity. Mixtures of unsolubilized fractions of the untreated bacteria with solubilized fractions of either the treated or untreated bacteria yielded reconstituted membranes with restored ATP-linked transhydrogenase activity. The level of the activity was similar to that of the undissociated membranes of untreated bacteria. The membranes which were reconstituted from unsolubilized fractions of the treated bacteria and the solubilized fraction of the treated or the untreated bacteria showed impairment of activity. The impairment is not due to an inability to bind ATPase of the soluble fraction or to an incorrect binding of the ATPase. The impaired, reconstituted membranes showed striking decreases in the relative amounts of three proteins with apparent molecular weights of 122,000, 73,000, and 62,000. The affected proteins were found to be components of the unsolubilized membrane fraction. It is, thus, concluded that the impaired activity is due to the defective nature of the unsolubilized membrane fraction of colicin-treated cells.     

Effect of colicin K on a membrane-associated, energy-linked function.

The purpose of this work was in investigate the capability of cell extracts of Escherichia coli and E. coli treated with colicin K to catalyze the following energy-dependent reverse transhydrogenase reaction: NADP + NADH + ATP in equilibrium NADPH + NAD +ADP + Pi. Under anaerobic conditions this reaction requires the presence of a specific portion of the electron transport chain, a functional energy coupling system, including an adenosine triphosphatase, enzyme, and ATP as energy source. The ATP-linked reaction was partially inhibited in French press extracts of E. coli K-12 C600 cells that had been pretreated with colicin K but not in extracts from similarly treated cells of a colicin-tolerant mutant. Ultracentrifugation of extracts yielded particulate fractions competent in catalyzing the reaction; this reaction is substantially inhibited in fractions from colicin-treated cells. The extent of inhibition increased with increasing concentration of colicin. Supernatants also supported ATP-linked formation of NADPH, but this reaction was insensitive to the colicin effect. A comparison between the requirement of the reaction in supernatant and particulate fractions suggests that the reaction in the supernatant is different from the one inhibited by colicin. The ATP-hydrolyzing ability of particulate fractions from the control or treated bacteria was identical. Likewise, the electron transport chain was not affected by colicin treatment, as evidenced from lack of effect on NADH oxidase, succinic dehydrogenase, and NADPH-NAD transhydrogenase. It is concluded that colicin K interferes with the coupling of ATP the utilization of the intermediate for the ATP-linked transdehydrogenase reaction.     

Studies of colicin action on wall-less stable L-forms of Escherichia coli. I. Degree of attachment and of killing effect on rods and stable L-form cells.

Escherichia coli strains B and K12 W 1655 F+ are able to bind more lethal units of colicins E2, E3, G, H, Ia, and K+ X per one stable L-form cell (of the protoplast type) than per one rod cell; colicin D is bound in a higher amount on E. coli B rods. This pattern remains unchanged, if the same colicins are attached on chloroform-killed cells of both forms. Rods of both E. coli strains are more sensitive to colicins D, E2, E3, K + X (as--in the strain B--to colicin Ia) than cells of the respective L-forms. In the strain W 1655 F+ both cell forms are equally highly sensitive to colicin Ia. The stable L-forms of both strains are much more sensitive to colicins G and H than the rods. Thus the Gram-negative cell wall decreases the probability of a colicin molecule to get attached to its receptor in the cytoplasmic membrane. On the other hand, in E. coli cells the attachment of most colicin molecules to the wall receptors increases the probability of their biological effect. There is no such effect of the wall-attachment on the action of colicins G or H. The strain B is tolerant to colicin E2, while being resistant to E3; thus the cytoplasmic membrane receptor sites for them are not identical.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.     

Mutant of Escherichia coli defective in response to colicin K and in active transport.

A mutant of Escherichia coli has been isolated that grows poorly on succinate and exhibits a markedly reduced sensitivity to colicin K. This mutant is also deficient in the respiration-linked transport of proline and thiomethyl-beta-D-galactoside but appears normal for the adenosine triphosphate-dependent transport of glutamine and arginine. A temperature-conditional revertant of the mutant grows on succinate and is sensitive to colicin K at 27 C, but fails to grow on succinate and is insensitive to colicin K at 42 C. Proline transport in the temperature-conditional revertant is reduced at 42 C when either glucose or succinate is used as energy source. Glutamine transport, on the other hand, is normal at 42 C with glucose as energy source, but is reduced with succinate, although not to the same extent as is proline transport. The lack of growth on succinate and the deficiencies in transport at 42 C are not due to a temperature-dependent lesion in either the electron transport chain or in Ca2+, Mg2+-activated adenosine triphosphatase activity. Membrane vesicles prepared from the temperature-conditional revertant are impaired in proline transport at both 27 and 42 C. These findings suggest the existence in the cytoplasmic membrane of E. coli cells of a component, presumably protein, that is required for colicin K action and that functions in respiration-linked and, to a lesser degree, in adenosine triphosphate-dependent active transport systems. This protein may serve as the primary target of colicin K action.     

Global structural rearrangement of the cell penetrating ribonuclease colicin E3 on interaction with phospholipid membranes

Nuclease type colicins and related bacteriocins possess the unprecedented ability to translocate an enzymatic polypeptide chain across the Gram-negative cell envelope. Here we use the rRNase domain of the cytotoxic ribonuclease colicin E3 to examine the structural changes on its interaction with the membrane. Using phospholipid vesicles as model membranes we show that anionic membranes destabilize the nuclease domain of the rRNase type colicin E3. Intrinsic tryptophan fluorescence and circular dichroism show that vesicles consisting of pure DOPA act as a powerful protein denaturant toward the rRNase domain, although this interaction can be entirely prevented by the addition of salt. Binding of E3 rRNase to DOPA vesicles is an endothermic process (DeltaH=24 kcal mol-1), reflecting unfolding of the protein. Consistent with this, binding of a highly destabilized mutant of the E3 rRNase to DOPA vesicles is exothermic. With mixed vesicles containing anionic and neutral phospholipids at a ratio of 1:3, set to mimic the charge of the Escherichia coli inner membrane, destabilization of E3 rRNase is lessened, although the melting temperature of the protein at pH 7.0 is greatly reduced from 50 degrees C to 30 degrees C. The interaction of E3 rRNase with 1:3 DOPA:DOPC vesicles is also highly dependent on both ionic strength and temperature. We discuss these results in terms of the likely interaction of the E3 rRNase and the related E9 DNase domains with the E. coli inner membrane and their subsequent translocation to the cell cytoplasm.     

Factors necessary for the export process of colicin E1 across cytoplasmic membrane of Escherichia coli

Factors necessary for the export process of colicin E1 across the cytoplasmic membrane of Escherichia coli were investigated. beta-Galactosidase activities from gene fusions between the colicin E1 and lacZ genes were recovered in the inner membrane fraction of E. coli when the region containing the internal signal-like sequence of colicin E1 [M. Yamada et al. (1982) Proc. Natl Acad. Sci. USA 79, 2827-2831] was present, but were found in the soluble fraction when the region was eliminated. The colicin E1 export was reduced upon insertion mutation in a gene that is located downstream from the colicin E1 gene in the same operon and responsible for mitomycin-C-induced killing of the host cell. A frame shift mutation of the colicin E1 plasmid was constructed to direct the protein which had lost the COOH-terminal 13 residues of original colicin E1 and was altered in 6 residues of the new COOH-terminal portion. The aberrant colicin E1 that was inducibly synthesized remained inside the cells. These results indicate that colicin E1 is exported with the aid of a product of the downstream gene and that the COOH-terminal portion is necessary for the export. The binding of colicin E1 to the cytoplasmic membrane through the internal signal-like sequence may be a step in the protein export process.     

Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli

The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied. In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein. E. coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA. Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release. This release was, however, still dependent on a functioning Cal protein. The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes. Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase. The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane. Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes. The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E. coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes.

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.     

Genetic Basis of Colicin E Susceptibility in Escherichia coli I. Isolation and Properties of Refractory Mutants and the Preliminary Mapping of Their Mutations

Colicin E-resistant mutants were isolated in Escherichia coli K-12 which, although still apparently possessing the E receptor and adsorbing colicin, were nevertheless insensitive (refractory) to its effect. Eight phenotypic groups were obtained, but some mutants from three of these groups were all shown to map at gal, whereas a second refractory locus, giving resistance to E1 alone, mapped close to thy. It is suggested that the successful fixation of any of the three distinct colicins of group E may involve a dual role for the cell surface "receptor," the first for the binding of the protein and the second for the correct orientation of the bound molecule relative to the cytoplasmic membrane. The majority of the refractory mutants isolated may derive from changes in components concerned with the second of these receptor functions. Two groups of mutants, however, refractory to only E1 or E2, probably reflect changes in the intracellular transmission systems which specifically mediate the effects of these two colicins, the changes not allowing transmission through the cytoplasmic membrane to the respective targets of the colicins. The E1 adsorption site was shown to be distinct from that for E2 and E3, indicating an early separation of the colicin E transmission systems.     

A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent Escherichia coli 113/3 cells

The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343-418. The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12-dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive.     

Translocation of an outer membrane protein into prey cytoplasmic membranes by bdellovibrios.

Within minutes of Bdellovibrio bacteriovorus attack on prey cells, such as Escherichia coli, the cytoplasmic membrane of the prey is altered. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified invaded prey cell (bdelloplast) membranes revealed the appearance of a noncytoplasmic membrane protein. This protein is not observed in preparations of noninvaded E. coli membranes and migrates in a manner similar to that of E. coli OmpF. Isoelectric focusing and two-dimensional gel electrophoresis of bdelloplast cytoplasmic membrane preparations also revealed the presence of a protein with electrophoretic properties similar to those of OmpF and the major Bdellovibrio outer membrane proteins. The protein appears in cytoplasmic membrane preparations within minutes of attack and persists throughout most of the intraperiplasmic developmental cycle. The appearance of this protein is consistent with our hypothesis that bdellovibrios translocate a pore protein into the bdelloplast cytoplasmic membrane to kill their prey and to gain access to the cytoplasmic contents for growth.     

The relation between the soluble factor associated with H(+)-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli.

Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H(+)-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Ths) and a membrane-bound component are together required for activity. Ths was selectively removed from chromatophore membranes of Rhs. rubrum and was purified to homogeneity by precipitation with (NH4)2SO4 and ion-exchange, affinity dye and gel exclusion chromatography. The latter indicated an Mr of approx. 74,000 under non-denaturing conditions but analysis of the pure protein by SDS-PAGE revealed a single polypeptide, Mr 43,000. Antibodies against this polypeptide inhibited transhydrogenase activity of chromatophores and decreased the capacity of Ths to restore activity to depleted membranes. They reacted with a polypeptide of Mr 43,000 in crude cell extract, chromatophore membranes and chromatophore washings but not with transhydrogenase polypeptides from the membranes of E. coli, Rb. capsulatus or animal mitochondria. The N-terminal amino acid sequence of the 43,000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the alpha subunit of the E. coli protein. The break between the alpha and beta polypeptides of E. coli transhydrogenase is such that both components are membrane-associated. In contrast, these results suggest that in the Rhs. rubrum enzyme Ths has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble. There is a predicted similarity between Ths and the reported sequence of alanine dehydrogenase from Bacillus but Ths did not have any alanine dehydrogenase activity.     

Purification and molecular properties of a new colicin.

The process of isolation and purification of a new colicin isolated from a Citrobacter strain is described. Escherichia coli sensitive cells are protected by vitamin B12 from the action of this bacteriocin; this suggests that it belongs to the E group of colicins. Therefore, we have called it colicin E4. It has a molecular weight of 56 000 and two molecular forms of isoelectric points 9.4 and 8.2 are separated in electrofocusing on polyacrylamide gels. It has a sedimentation coefficient of 3.4 S and the absorption coefficient A1(280%) nm is 6.23 cm(-1). Using an antibody raised against pure colicin E4, no cross-reaction was detected against colicins A, E1 or K. The physiological effect of colicin E4 on sensitive cells is very similar to that of colicins E1, K or I which disrupt the energized membrane state.     

Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F(1)F(o) ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.     

Acidic interaction of the colicin A pore-forming domain with model membranes of Escherichia coli lipids results in a large perturbation of acyl chain order and stabilization of the bilayer.

2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phototoxicity of phenothiazinium-based photosensitizers to bacterial membranes

The ability of phenothiazinium-based photosensitizers to induce photodamage to Escherichia coli membranes is investigated. Phenothiazinium-based photosensitizers were found to be somewhat lipophilic (log P>0.7) and to induce surface-pressure changes (3-12 mN m(-1)) in lipid monolayers mimetic of bacterial membranes, implying that these molecules are able to penetrate biological membranes. Under dark and light conditions (3.15 J cm(-1) for 30 min), phenothiazinium-based photosensitizers were incubated with E. coli cells. These cells showed levels of dark bacteriolysis that ranged between 6% and 13%, with light conditions leading to no significant increase in these levels. Gas chromatography-based analyses showed such incubations to produce no significant changes in the levels of C(16) and C(18) fatty acid chain saturation found in E. coli whole lipid-extracts. It is concluded that the phenothiazinium-based photosensitizers studied may not use E. coli membranes as their primary photodynamic target, but may inflict photodamage on cytoplasmic targets, possibly DNA.     

Structure and dynamics of the colicin E1 channel

The toxin-like and bactericidal colicin E1 molecule is of interest for problems of toxin action, polypeptide translocation across membranes, voltage-gated channels, and receptor function. Colicin E1 binds to a receptor in the outer membrane and is translocated across the cell envelope to the inner membrane. Import of the colicin channel-forming domain into the inner membrane involves a translocation-competent intermediate state and a membrane potential-dependent movement of one third to one half of the channel peptide into the membrane bilayer. The voltage-gated channel has a conductance sufficiently large to depolarize the Escherichia coli cytoplasmic membrane. Amino acid residues that affect the channel ion selectivity have been identified by site-directed mutagenesis. The colicin E1 channel is one of a few membrane proteins whose secondary structures in the membrane, predominantly alpha-helix, have been determined by physico-chemical techniques. Hypothesis for the identity of the trans-membrane helices, and the mechanism of binding to the membrane, are influenced by the solved crystal structure of the soluble colicin A channel peptide. The protective action of immunity protein is a unique aspect of the colicin problem, and information has been obtained, by genetic techniques, about the probable membrane topography of the imm gene product.