Alexandre Ghazi

In Vitro Multimerization and Membrane Insertion of Bacterial Outer Membrane Secretin PulD

Synthesis of the Klebsiella oxytoca outer membrane secretin PulD, or its membrane-associated core domain, in a liposome-supplemented Escherichia coli in vitro transcription-translation system resulted in the formation of multimers that appeared as typical dodecameric secretin rings when examined by negative-stain electron microscopy. Cryo-electron microscopy of unstained liposomes and differential extraction by urea indicated that the secretin particles were inserted into the liposome membranes. When made in the presence of the detergent Brij-35, PulD and the core domain were synthesized as monomers. Both proteins caused almost immediate growth cessation when synthesized in E. coli without a signal peptide. The small amounts of PulD synthesized before cell death appeared as multimers with characteristics similar to those of the normal outer membrane secretin dodecamers. It was concluded that multimerization and membrane insertion are intrinsic properties of secretin PulD that are independent of a specific membrane environment or membrane-associated factors. The closely related Erwinia chrysanthemi secretin OutD behaved similarly to PulD in all assays, but the more distantly related Neisseria meningitidis secretin PilQ did not form multimers either when made in vitro in the presence of liposomes or when made in E. coli without its signal peptide. This is the first report of the apparently spontaneous in vitro assembly and membrane insertion of a large outer membrane protein complex. Spontaneous multimerization and insertion appear to be restricted to outer membrane proteins closely related to PulD.

Excretion of glutamate from Corynebacterium glutamicum triggered by amine surfactants

Corynebacterium glutamicum is used for the industrial production of glutamate. Excretion of the amino acid may be induced by various means. We have analyzed the characteristics of glutamate excretion induced by two amine surfactants, dodecylammonium acetate (DA) and dodecyltrimethylammonium bromide (DTA). Addition of these surfactants induced an immediate efflux of internal glutamate. It also induced a perturbation of the energetic parameters of the cell (decrease of delta mu H, decrease of the internal ATP concentration). The efflux was not the result of these perturbations: glutamate is taken up by the cells via an ATP-dependent unidirectional active transport system and no efflux took place as a consequence of an artificial decrease of the energetic parameters. In addition, amine surfactants also induced an excretion of other species, in particular potassium. We have tested the possibility that the effluxes result from a permeabilization of the lipid bilayer by analyzing the interactions between the surfactants and liposomes.

Probes of membrane potential in Escherichia coli cells

Membrane potential (A

Lactose transport in Escherichia coli cells. Dependence of kinetic parameters on the transmembrane electrical potential difference.

) of cells which are too small to allow the use of microelectrodes must be indirectly determined. In bacterial cells, labelled permeant cations which distribute across the cell membrane in response to a potential difference (negative inside) are currently used [l]. At thermodynamic equilibrium, the membrane potential is related to the concentration of labelled monovalent ion C’ inside and outside the cell by the Nernst equation:

Lactose transport in Escherichia coli cells: Evidence in favor of a permease-catalyzed efflux of lactose without protons

We determine the kinetic parameters V and KT of lactose transport in Escherichia coli cells as a function of the electrical potential difference (delta psi) at pH 7.3 and delta pH = 0. We report that transport occurs simultaneously via two components: a component which exhibits a high KT (larger than 10 mM) and whose contribution is independent of delta psi, a component which exhibits a low KT independent of delta psi (0.5 mM) but whose V increases drastically with increasing delta psi. We associate these components of lactose transport with facilitated diffusion and active transport, respectively. We analyze the dependence upon delta psi of KT and V of the active transport component in terms of a mathematical kinetic model developed by Geck and Heinz (Geck, P. and Heinz, E. (1976) Biochim. Biophys. Acta 443, 49-63). We show that within the framework of this model, the analysis of our data indicates that active transport of lactose takes place with a H+/lactose stoichiometry greater than 1, and that the lac carrier in the absence of bound solutes (lactose and proton(s) is electrically neutral. On the other hand, our data relative to facilitated diffusion tend to indicate that lactose transport via this mechanism is accompanied by a H+/lactose stoichiometry smaller than that of active transport. We discuss various implications which result from the existence of H+/lactose stoichiometry different for active transport and facilitated diffusion.

Differences in uncoupling effects associated with the uptake of lactose and dansyl-galactoside in Escherichia coli membrane: Active transport versus specific binding

The mechanism of fl-galactoside transport in E. coli is a proton symport : the membrane-located carrier (lactose permease) cotransports a proton(s) and a molecule of lactose [ 1,2]. The entry of protons under an electrochemical potential difference across the cytoplasmic membrane is the exergonic process which drives the endergonic process, the accumulation of lactose. The original hypothesis was that the lactose permease catalyzed the reaction:

Implication of proteases in the respiration dependent inactivation of the lactose permease of E. coli

Abstract Cytoplasmic membrane vesicles isolated from Escherichia coli take up dansyl-galactoside, a fluorescent competitive inhibitor of lactose transport, to much lower levels than lactose. An initial interpretation, based on the study of the fluorescent changes accompanying the energy-dependent uptake, was that it represented a one-to-one specific binding to the lac carrier protein which was not followed by transport. Recently, on the basis of a new estimation of the number of lac carrier in the membrane, it has been advanced that the uptake of dansyl-galactoside represents a nonspecific binding on the inner surface of the membrane following transport. We discriminate between the two interpretations by comparing the effects of lactose and dansyl-galactoside uptake on the electrochemical gradient of protons ( Δ \ gm H + ), generated by the oxidation of substrates, and on the uptake of proline. Indeed, it is known that the rate of lactose transport is such that it leads, as a consequence of the lactose/H + symport, to an observable decrease of Δ \ gm H + , and secondary to this decrease to an inhibition of the uptake of proline transported at much lower rate. We show that the rates of uptake of lactose and dansyl-galactoside by the membrane vesicles are similar; yet the uptake of dansyl-galactoside does not lead to the uncoupling effects which are associated with the uptake of lactose. We discuss the possible reasons for the absence of this uncoupling effect, and we conclude that our data are incompatible with the notion that the energy-dependent uptake of dansyl-galactoside is associated with an active transport involving a dansyl-galactoside/H + symport. On the contrary, the data substantiate the initial interpretation that the energy-dependent uptake of dansyl-galactoside reflects the binding to the lac carrier not followed by transport.

Inactivation of the lactose permease of Escherichia coli by the respiratory activity

The lactose permease of E. coli becomes irreversibly inactivated during lactose transport under conditions of high respiratory activity. This inactivation is characterized by a decrease in the steady state of lactose accumulation, a decrease in the influx rate of lactose, and a decrease in the transmembrane electrical potential. We report here that inhibitors of serine proteases (phenylmethylsulfonyl fluoride and N‐α‐P‐tosyl‐L‐lysine chloromethyl ketone) prevent this inactivation, thus implicating proteases in this process.

Absence of a unique relationship between active transport of lactose and protonmotive force in E. coli

Abstract We had shown previously that the lactose permease of Escherichia coli ML 308225 becomes irreversibly inactivated during lactose transport when the cells are energized by addition of an external energy source; this inactivation is concomitant with an irreversible decrease of the electrochemical potential gradient of protons (Ghazi, A., Therisod, H. and Shechter, E. (1983) J. Bacteriol. 154, 92–103). Addition to energized cells of 2-heptyl-4-hydroxyquinoline- N -oxide (HQNO), an inhibitor of the respiratory chain, suppresses these phenomena. Also, the inactivation of the lactose permease does not take place in energized E. coli K 207 cells, a mutant devoid of a functional respiratory chain. The inactivation of the lactose permease may take place in nonenergized cells and in the absence of lactose, but at a much slower rate. Addition of lactose or methyl-1-thio-β- d -galactoside (TMG), a competitive analogue of lactose, enhances the inactivation. On the other hand, addition of β- d -galactosyl-1-thio-β- d -galactoside (TDG), or p- nitrophenyl -α- d -galactoside (α-NPG), or o- nitrophenyl -β- d -galactoside (ONPG), other competitive analogues of lactose, strongly inhibits the inactivation. From these data, it is concluded that the respiratory activity of the cell in itself leads to an inactivation of the lactose permease. The presence of one class of galactosides enhances the susceptibility of the permease towards inactivation, probably by immobilizing the protein in a conformation more susceptible to the inactivating agent. In contrast, the presence of another class of galactosides can protect the protein against inactivation.

The Lactose Carrier of E. Coli: Protease Dependent in Vivo Inactivation

The relationship between active transport of lactose via the lactose permease and the protonmotive force has been determined in E. coli cells using either the respiratory chain inhibitor cyanide or protonophores to decrease the protonmotive force progressively. In contradiction with the prediction of the delocalized chemiosmotic theory, two different relationships were obtained depending on the method used.