Mode of action of colicin ib: formation of ion-permeable membrane channels

Addition of purified colicin Ib to whole Escherichia coli cells or cytoplasmic membrane vesicles inhibits their subsequent ability to generate a membrane potential. In addition, this colicin is shown to bring about a voltage-dependent increase in the conductance of an artificial planar bilayer membrane prepared from soybean phospholipids. This results from the formation of ion-permeable channels. These data provide strong evidence that the depolarization of Escherichia coli cells by this colicin results from an Ib-induced increase in membrane permeability to ions.     

Permeability to cobalt ions and action of colicin K in a mutant that overproduces cardiolipin

A mutant of Escherichia coli that produces excess cardiolipin becomes less capable of transporting Co²⁺. Cardiolipin therefore does not act as an ionophore under these conditions. Colicin K brings about the typical increase in permeability to Co²⁺ in the mutant.     

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na⁺ gradient ([$\text{Na}{}^{\mathrm{+}}\text{]}\text{outside}\text{> [}\text{Na}{}^{\mathrm{+}}\text{]}\text{inside}$). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$ and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.     

Light-driven proton translocations in Halobacterium halobium

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating an electrochemical proton gradient across the cell membrane. However, the typical response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light is turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium.The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow.The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N′-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor.Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Requirement of heat and metabolic energy for the expression of inhibitory action of colicin K

Escherichia coli B, induced for β-galactoside permease, can accumulate thiomethyl-β-galactoside in the cell even at 0 °C. At this temperature, cells adsorb colicin K but the adsorbed colicin does not inhibit thiomethyl-β-galactoside uptake. Inhibition by colicin K is, however, seen at 0 °C after exposure of the colicin K-cell complex to a high temperature: a greater degree of inhibition occurs with increasing temperature or duration of exposure. There is a transition point at around 21 °C in Arrhenius plots of this colicin K activation reaction.If inhibitors of energy yielding reactions are present during the heat treatment, the inhibitory action of colicin K (as measured by thiomethyl-β-galactoside uptake after returning the colicin K-cell complex to 0 °C and removal of the inhibitors) is prevented.These results indicate that adsorbed colicin K is converted into the active state only in the presence of metabolic energy and that cell surface fluidity appears to be concerned in this process.     

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.     

Formation of large,ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order of 10 ng/cm³ to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS ($\text{1}\text{nS}\text{= 10}{}^{\mathrm{?9}}\text{siemens}\text{= 10}{}^{\mathrm{?9}}\text{Ω}{}^{\mathrm{?1}}$) in 1 M KCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.     

Enterotoxin of Clostridium perfringens type A forms ion-permeable channels in a lipid bilayer membrane

The enterotoxin of Clostridium perfringens type A was found to form ion-permeable channels in a lipid bilayer. A patch clamp technique was used to detect channel activities in an asolectin bilayer with incorporated enterotoxin. About 20% of the lipid bilayer patches examined showed rectangular or stepwise shift of membrane current. The shifts indicated the gating of ion-permeable channels in the patches. The channels showed high conductance (40-450 pS), no rectification in current-voltage curves and occasional long-lasting events. The significance of these findings is discussed in relation to the mechanism of action of the toxin.     

Isolation of conjugation-constitutive mutants of colicin factor Ib

Summary Colicin factor ColIb-P9 is known to act as a sex factor in E. coli or Salmonella. Although ColIb-P9 confers mating ability on its host bacteria, this ability appears to be repressed since only a small proportion of cells in a culture of a colicinogenic strain are able to pair with, and transmit the factor to recipient bacteria. We have isolated mutants of ColIb-P9 which confer constitutive donor ability on their host. De-repression in these mutants is probably due to failure to produce repressor, rather than to insensitivity to repressor. As the colicin production by the mutants is still repressed, colicin synthesis and conjugation ability are subject to independent systems of regulation.     

Mechanosensitive membrane channels in action

The tension-driven gating process of MscL from Mycobacterium tuberculosis, Tb-MscL, has been addressed at near-atomic detail using coarse-grained molecular dynamics simulations. To perform the simulations, a novel coarse-grained peptide model based on a thermodynamic parameterization of the amino-acid side chains has been applied. Both the wild-type Tb-MscL and its gain-of-function mutant V21D embedded in a solvated lipid bilayer have been studied. To mimic hypoosmotic shock conditions, simulations were performed at increasing levels of membrane tension approaching the rupture threshold of the lipid bilayer. Both the wild-type and the mutant channel are found to undergo significant conformational changes in accordance with an irislike expansion mechanism, reaching a conducting state on a microsecond timescale. The most pronounced expansion of the pore has been observed for the V21D mutant, which is consistent with the experimentally shown gain-of-function phenotype of the V21D mutant.     

Restriction endonuclease mapping of the colicin Ib plasmid

Summary A cleavage site map of the colicin Ib plasmid (ColIb) has been determined for the enzymes Sall, XhoI, and HindIII by analysis of partial digests, double digests, DNA-DNA hybridization, and Tn5-induced insertion mutants. The site of the colicin gene has been determined by probing with cloned DNA coding for colicin production, as well as by analysis of a colicin negative ColIb:Tn5.     

The dynamic activation of colicin Ia channels in planar bilayer lipid membrane

Dynamic activation of ion channels formed by colicin Ia incorporated into a planar bilayer lipid membrane (BLM) was investigated by the voltage clamp technique using different step voltage stimuli. We have demonstrated a critical resting interval, Deltat(c), between two identical successive voltage pulses. If the second pulse is applied within Deltat(c), it produces a predictable current response. On the contrary, if the second pulse is applied after Deltat(c), the current response cannot be reliably predicted. Computer simulations based on an idealized mathematical model, developed in this paper, qualitatively reproduce the system's dynamic responses to stimulus trains. The behavior of the ion channels, when the resting period exceeds Deltat(c), may be interpreted as a transient gain or loss or resetting of memory, as revealed by a specific sequence of electrical pulses used for stimulation.