Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli

Osmotic upshock of E. coli cells in NaCl or sucrose medium resulted in a large decrease in the cytoplasmic volume and the inhibition of growth, of the electron transfer chain and of four different types of sugar transport system: the lactose proton symport, the glucose phosphotransferase system, the binding-protein dependent maltose transport system and the glycerol facilitator. In contrast to NaCl and sucrose, the permeant solute glycerol had no marked effect. These inhibitions could be partially relieved by glycine betaine. Despite these inhibitions, the internal pH, the protonmotive force and the ATP pool were maintained. It is concluded that inhibition of electron transfer and of sugar transport is the consequence of conformational changes caused by the deformation of the membrane. It is also concluded that the arrest of growth observed upon osmotic upshock is not due to energy limitations and that it cannot be explained by the inhibition of carbohydrate transport.     

Effects of ethanol on the Escherichia coli plasma membrane.

The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids. The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes. Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface. In comparison, hexanol decreased the polarization of probes located more deeply in the membrane. Temperature had a large effect on probes located at all depths. The effects of ethanol on E. coli membranes from cells grown with or without ethanol were also examined. Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells. In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells. These differences are explained by analyses of membrane composition. Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio. This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth. Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E. coli to growth in the presence of ethanol.     

Photolyase-dimer-DNA complexes and exclusion stimulation in Escherichia coli: Depolarization of the plasma membrane

Using cells that overproduce DNA photolyase, we found that UV irradiation (3 J/m²) efficiently inactivates accumulation of methylthiogalactoside (TMG) when RexAB proteins of phage lambda are present. The effect requires both formation of photolyase-dimer-DNA (PDD) complexes and expression of the RexAB proteins. It is reversed completely by a flash of visible light if given immediately after UV and becomes irreversible after post-UV incubation for about 15 min. Inactivation is significant after only 5 min of post-UV incubation, is accompanied by a loss of previously accumulated TMG, and does not require de novo protein synthesis. Passive transport of O-nitrophenylgalactoside by inactivated cells is typical of energy-depleted membranes. We suggest that PDD complexes mimic a developmental intermediate of phage superinfection and stimulate formation of the RexB membrane channel recently proposed by others to explain classical “exclusion”. This suggestion is supported by additional data showing an inactivation of colony-forming ability by exclusion stimulation and an inability of PDD complexes to inactivate accumulation of TMG if RexB is present in larger relative amounts than RexA (a detail characteristic of exclusion stimulated by phage superinfection).     

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress

Aims: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. Methods and Results: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55–70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE? system to measure net K⁺ fluxes. No difference in K⁺ fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K⁺ loss from the heat‐treated E. coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K⁺ flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K⁺ flux disruption was not clearly observed until 68°C was reached. Conclusions: No changes in net K⁺ flux from heat‐stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K⁺ fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two‐stage process enabled evaluation of the lethality of heat‐treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. Significance and Impact of the Study: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture‐dependent test offers an alternative system to culture‐based methods.     

Viability and virulençe of Escherichia coli suspended by membrane chamber in semitropical ocean water

Abstract Escherichia coli H10407 was suspended in seawater (38.5‰ salinity) contained in membrane chambers (0.4-μm polycarbonate membrane) incubated in situ at 25°C in Nixon's Harbor, South Bimini, Bahamas. Although colonies of E. coli could not be cultured after 13 h post chamber inoculation, the number of fluorescent-antibody staining cells remained constant. Direct viable counts revealed that viable cells were present, even though the cell suspension was not culturable on the media tested. After exposure to seawater for 112 h, cells were concentrated by centrifugation and introduced into ligated rabbit ileal loops. E. coli H10407 proved viable for recovery from inoculated loops and was confirmed by detection of characteristic plasmid bands. Results indicate that enteric pathogens remain viable in seawater long after they cease to be cultivable on laboratory media.     

Cloning and expression of the yeast plasma membrane ATPase in Escherichia coli

The yeast plasma membrane ATPase gene PMA1 was cloned into Escherichia coli using the high expression tac and T7 promoters. The gene product is toxic to the bacterial cell leading to very low expression levels and arrested growth of the host cell within minutes of induction. The expressed protein is immunologically cross-reactive with the yeast ATPase, comigrates with the original protein in sodium dodecyl sulfate-polyacrylamide gels, and is isolated in the E. coli membrane fraction. The partially purified protein exhibits ATPase activity.     

Glycine betaine transport in Escherichia coli: osmotic modulation.

Exogenous glycine betaine highly stimulates the growth rate of various members of the Enterobacteriaceae, including Escherichia coli, in media with high salt concentrations (D. Le Rudulier and L. Bouillard, Appl. Environ. Microbiol. 46:152-159, 1983). In a nitrogen- and carbon-free medium, glycine betaine did not support the growth of E. coli either on low-salt or high-salt media. This molecule was taken up by the cells but was not catabolized. High levels of glycine betaine transport occurred when the cells were grown in media of elevated osmotic strength, whereas relatively low activity was found when the cells were grown in minimal medium. A variety of electrolytes, such as NaCl, KCl, NaH2PO4, K2HPO4, K2SO4, and nonelectrolytes like sucrose, raffinose, and inositol triggered the uptake of glycine betaine. Furthermore, in cells subjected to a sudden osmotic upshock, glycine betaine uptake showed a sixfold stimulation 30 min after the addition of NaCl. Part of this stimulation might be a consequence of protein synthesis. The transport of glycine betaine was energy dependent and occurred against a concentration gradient. 2,4-Dinitrophenol almost totally abolished the glycine betaine uptake. Azide and arsenate exerted only a small inhibition. In addition, N,N'-dicyclohexylcarbodiimide had a very low inhibitory effect at 1 mM. These results indicated that glycine betaine transport is driven by the electrochemical proton gradient. The kinetics of glycine betaine entry followed the Michaelis-Menten relationship, yielding a Km of 35 microM and a Vmax of 42 nmol min-1 mg of protein-1. Glycine betaine transport showed considerable structural specificity. The only potent competitor was proline betaine when added to the assay mixtures at 20-fold the glycine betaine concentration. From these results, it is proposed that E. coli possesses an active and specific glycine betaine transport system which is regulated by the osmotic strength of the growth medium.     

Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli

Fluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer‐Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane‐spanning alpha‐helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature‐dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.     

Escherichia coli and Lactobacillus plantarum responses to osmotic stress

Escherichia coli and Lactobacillus plantarum were subjected to final water potentials of −5.6 MPa and −11.5 MPa with three solutes: glycerol, sorbitol and NaCl. The water potential decrease was realized either rapidly (osmotic shock) or slowly (20 min) and a difference in cell viability between these conditions was only observed when the solute was NaCl. The cell mortality during osmotic shocks induced by NaCl cannot be explained by a critical volume decrease or by the intensity of the water flow across the cell membrane. When the osmotic stress is realized with NaCl as the solute, in a medium in which osmoregulation cannot take place, the application of a slow decrease in water potential resulted in the significant maintenance of cell viability (about 70–90%) with regard to the corresponding viability observed after a sudden step change to same final water potential (14–40%). This viability difference can be explained by the existence of a critical internal free Na⁺ concentration. Received: 20 May 1998 / Received revision: 31 July 1998 / Accepted: 31 July 1998     

Preprotein translocation creates a halide anion permeability in the Escherichia coli plasma membrane.

The electrochemical potential drives the translocation of the precursor form of outer membrane protein A (proOmpA) and other proteins across the plasma membrane of Escherichia coli. We have measured the electrical potential, delta psi, across inverted membrane vesicles during proOmpA translocation. delta psi, generated by the electron transport chain, is substantially dissipated by proOmpA translocation. delta psi dissipation requires SecA, ATP, and proOmpA. proOmpA which, due to the covalent addition of a folded protein to a cysteinyl side chain, is arrested during its translocation, can nevertheless cause the loss of delta psi. Thus the movement of charged amino acyl residues is not dissipating the potential. This translocation-specific reduction in delta psi is only seen in the presence of halide anions, although halide anions are not needed for proOmpA translocation per se. We therefore propose that translocation intermediates directly increase the membrane permeability to halide anions.     

Effect of acriflavine on the plasma membrane of Escherichia coli K12

Plasma membranes of acriflavine-sensitive mutant (acrA) and acriflavine-resistant (acrA+, wild-type and true revertant) Escherichia coli K12 strains treated with acriflavine were observed under the electron microscope by means of the freeze-fracture technique. The plasma membrane of the acrA mutant exhibited a complex lamellar structure at the end of the cell when treated with 20 micrograms acriflavine ml-1. However, the membrane of the acrA+ cells also gave the lamellar complex when treated with a very high concentration of acriflavine (100 micrograms ml-1). The size of the intramembranous particles was not affected by the acriflavine treatments.     

Characterization of Escherichia coli nucleoids released by osmotic shock

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 μg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5-10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (<2 s) was on average ～42 μm(3). Lysozyme at concentrations above 1 μg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 μg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 μM ethidium bromide, but not with chloroquine. While DNase (1 μg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 μg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network.     

Topographical features of the membrane of Poterioochromonas malhamensis after colchicine and osmotic treatment

Changes in membrane topography in the flagellate Poterioochromonas malhamensis, as a result of colchicine and osmotic-stress treatments, have been studied using freeze-fracturing and thin sectioning. Ridges, but not rows of intramembrane particles, in the PF-face which denote the position of underlying cortical microtubules, together with the ridge associated with their point of origin (flagellar root fibre 1), dissappear after colchicine or short-term (5 min) osmotic treatments. Cortical microtubules are destroyed as a result of the former, but not the latter treatment. Longer periods in osmoticum allow a recovery of the microtubule — associated membrane ridges. Despite careful isosmotic fixations distinct cross-bridges between microtubules and the plasmalemma were not discernible in thin section.     

Protein mobility and diffusive barriers in Escherichia coli: consequences of osmotic stress

The effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo-bleaching, pulsed-FRAP. This method employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed-FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion (D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 microm(2) s(-1), whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high (D approximately 0.4 microm(2) s(-1)). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed.     

Escherichia coli membrane proton conductance and proton efflux depend on growth pH and are sensitive to osmotic stress

The dependence of Escherichia coli membrane H+ conductance (Gm H+) with a steady-state pH in the presence and absence of an external source of energy (glucose) was studied, when cells were grown under anaerobic and aerobic conditions, with an assay pH of 7.0. Energy-dependent H+ efflux by intact cells growing at pH of 4.5-7.5 was also measured. The elevated H+ conductance and lowered H+ flux were shown for cells growing in acidic pH and under anaerobic conditions, when bacteria were fermenting glucose. The atp mutant, which is deprived of the F0F1- adenosine triphosphatase, had less Gm H+ independent of growth conditions. In contrast with wild-type or precursor strain, a remarkable difference in Gm H+ for atp mutant was observed between aerobic and anaerobic conditions; such a difference was significant at pH 4.5. These results could indicate distinguishing pathways determining Gm H+ under anaerobic conditions after the fermentation of glucose at different pH and an input of the F0F1-adenosine triphosphatase in Gm H+. In addition, the effect of osmotic stress was demonstrated with grown cells. Gm H+ and H+ efflux both were increased after hyperosmotic stress at pH 7.5, and these changes were inhibited by N,N\'-dicyclohexylcarbodiimide, whereas these changes were lower in atp mutant. A role of the F0F1-adenosine triphosphatase in osmo-sensitivity of bacteria was confirmed under fermentative conditions.     

Location and mobility of twin arginine translocase subunits in the Escherichia coli plasma membrane

The twin arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Two primary Tat complexes have been identified, comprising TatABC or TatA multimers, which may interact at the point of translocation. We have analyzed green/cyan/yellow fluorescent protein (XFP) fusions to each of the Tat subunits. We show that the TatB and TatC fusions are active and incorporated into purified TatABC complexes. Proteolytic clipping of the TatA-XFP fusion precludes a definitive conclusion regarding activity, but we do find that the full fusion protein is preferentially incorporated into the TatABC complex. A previous study has proposed that TatB and possibly TatC are localized at the cell poles, whereas TatA is distributed more uniformly throughout the plasma membrane. Here, we likewise show that TatA-XFP is primarily distributed around the periphery of the cell. However, whereas much of the TatB-XFP is found at the poles, quantitative imaging studies show that approximately half of the protein is uniformly distributed in the plasma membrane. Moreover, we show that the bulk of TatC-XFP is detected as a halo around the cells, in some cases as punctate areas that are much smaller than those occupied by TatB-green fluorescent protein (GFP), indicating a uniform distribution. No evidence for a polar localization of TatC-GFP was obtained. Although TatC-GFP is found correctly complexed with TatB, a high proportion of TatB-GFP is not linked to TatC, and we propose that this "free" TatB forms unphysiological assemblies, possibly because it is synthesized in excess. Since TatC is invariably complexed with TatB in wild-type complexes, the combined data demonstrate that TatABC complexes are uniformly distributed throughout the plasma membrane. The significance of the punctate TatA/B/C-GFP is unclear; fluorescence recovery after photobleaching measurements show that these pools of proteins are immobile, whereas nonaggregated proteins are highly mobile in the plasma membrane.     

Stoicheiometry of lactose–proton symport across the plasma membrane of Escherichia coli

The rate of [(14)C]lactose inflow into anaerobic, non-metabolizing, Escherichia coli has been correlated with the rate of the simultaneous effective inflow of H(+). It is shown that the initial rates of the two transport processes are essentially the same, and it is concluded that the two processes are strictly coupled with a 1:1 stoicheiometry.     

Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli.

Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane.