Emanuel Shechter

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-delta-9-octadecenoate, trans-delta-9-hexadecenoate or cis-delta-9-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, Tt, and the range of the transition, delta-T, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60-80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25-40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.

The S‐layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C‐terminal hydrophobic domain

PS2 is the S‐layer protein of Corynebacterium glutamicum. The S‐layer may be detached from the cell as organized sheets by detergents at room temperature. The solubilization of PS2 in the form of monomers requires detergent treatment at high temperature (70°C), conditions under which the protein is denatured. Treatment of the cells with proteinase K or trypsin results in the detachment of the organized S‐layer, which remains organized. Because we show that trypsin cleaves the C‐terminal part of the protein, we conclude that this domain is involved in the association of the S‐layer to the cell but is not essential in the interaction between individual PS2 proteins within the S‐layer. A modified form of PS2, deleted of its C‐terminal hydrophobic sequence, was constructed. The protein is almost unable to form an organized S‐layer and is mainly released into the medium. We suggest that PS2 is anchored via its C‐terminal hydrophobic sequence to a hydrophobic layer of the wall of the bacterium located some distance above the cytoplasmic membrane.

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.

Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: A combined freeze-etch electron microscopy and biochemical study

Summary— The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze‐fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimension as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced the ordered fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer.

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

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.

Fluorescence dye as monitor of internal pH in Escherichia coli cells

The most widely used techniques for the determination of intracellular pH are based upon the distribution of an isotopically labeled or fluorescent weak acid or weak base between the intracellular space and the external medium [ 1,2]. While this technique is direct, it is not always the method of choice [3], particularly for rapid or transient pH changes where equilibration time can be limiting and/or the need to isolate the cells from their medium before each measurement constitutes a time problem. Measurement of intracellular pH via 3’P nuclear magnetic resonance of the pH-dependent chemical shift [4-61 is not always applicable to rapidly changing systems. This is a modification of the original technique allowing the determination of such alkaline intracellular pHi. It uses the strong pH-dependence of the rate of intracellular hydrolysis of 6_carboxyfluorescein-diacetate by non-specific esterases. We have applied this technique to the determination of pHi of E. coli cells, which is known to be alkaline [4,13,14].

The lactose permease of Escherichia coli: Evidence in favor of a dimer

Lactose permease from Escherichia coli T 206 was purified in octyl-beta-D-glucopyranoside (octyl-glucoside) according to Newman et al. [J. Biol. Chem. (1981) 256, 11804-11808]. In this detergent the protein has a very high tendency to aggregate nonspecifically. Therefore, exchange of octyl-glucoside was performed for another nonionic detergent, dodecyl octaethylene glycol monoether (C12E8), in which the protein is more stable. The amounts of bound C12E8 and phospholipids were measured using radioactive detergent and gas chromatography, respectively, and were found to be respectively 0.2 and 0.15 g/g protein. Analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium) and gel filtration (conventional and high performance liquid chromatography) experiments indicated that in this detergent the lactose permease existed mainly as a dimer. This result is at variance with the monomeric state of the protein reported by Wright et al. [FEBS Lett. (1983) 162, 11-15] in another nonionic detergent (dodecyl-o-beta-maltoside). We discuss the possible reason for this discrepancy and suggest that the dimeric state of association may well reflect the situation that prevails in the membrane.

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

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:

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

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.

Characterization of energetically functional inverted membrane vesicles from Corynebacterium glutamicum

We show that inverted membrane vesicles from Corynebacteriwn glutamicum, a Gram‐positive bacterium, are able to generate and maintain an electrochemical gradient of protons in response to the addition of NADH. This result indicates that the respiratory chain is intact and that the vesicles are reasonably impermeable to protons. These membrane vesicles may be the starting point for in vitro translocation studies of proteins in Gram‐positive bacteria.