Diffusion of green fluorescent protein in three cell environments in Escherichia coli

Surprisingly little is known about the physical environment inside a prokaryotic cell. Knowledge of the rates at which proteins and other cell components can diffuse is crucial for the understanding of a cell as a physical system. There have been numerous measurements of diffusion coefficients in eukaryotic cells by using fluorescence recovery after photobleaching (FRAP) and related techniques. Much less information is available about diffusion coefficients in prokaryotic cells, which differ from eukaryotic cells in a number of significant respects. We have used FRAP to observe the diffusion of green fluorescent protein (GFP) in cells of Escherichia coli elongated by growth in the presence of cephalexin. GFP was expressed in the cytoplasm, exported into the periplasm using the twin-arginine translocation (Tat) system, or fused to an integral plasma membrane protein (TatA). We show that TatA-GFP diffuses in the plasma membrane with a diffusion coefficient comparable to that of a typical eukaryotic membrane protein. A previous report showed a very low rate of protein diffusion in the E. coli periplasm. However, we measured a GFP diffusion coefficient only slightly smaller in the periplasm than that in the cytoplasm, showing that both cell compartments are relatively fluid environments.     

Cellular spin resonance: Theory and experiment

Living and dead cells, as well as certain inanimate particles, can be made to spin in a rotating electric field, such as that produced by a four-pole electrode system. The theory for the effects and the experimental results for a number of cells are given. Yeast cells (Saccharomyces cerevisiae, dead, and as protoplasts;Schizosaccharomyces octospora); an alga (Chlorella pyrenoidosa); mouse myeloma cells; human Hela cells; and several model particles were studied. Their spectra of spin response are shown to agree with the theory presented. Interestingly, live cells spin in a contrafield direction, while dead ones spin co-field in the low-frequency range. The result indicate that this new technique of cellular spin resonance (CSR) will be useful in determining the effective dielectric constant of individual cells, for the direction and magnitude of the CSR is directly proportional to the effective dielectric constant, and the spin rate can readily and directly be determined over a wide frequency range.     

Restricted diffusion in biophysical systems: Theory

Theoretical results are presented on measurements of restricted diffusion in biophysical systems by the pulsed gradient spin echo nuclear magnetic resonance (PGSENMR) technique. A Fokker-Planck equation is developed to describe restricted diffusion, and it is shown that only two basic types of penetrable diffusion barriers exist, those in which the diffusing particles are partially excluded from the barrier region because of an increased free energy, and those in which the diffusing particles are not excluded but experience increased viscosity in the region. The Fokker-Planck equation is used to obtain expressions for the spin echo amplitude in PGSENMR experiments, and it is shown that for restricted diffusion the average diffusion coefficient measured in these experiments over short intervals is larger than that measured over long intervals. The possibility of distinguishing between the two types of barriers is considered. The experimental parameters required for intracellular restricted diffusion measurements are discussed, and it is shown that the interpretation of PGSENMR results in animal tissues should include the possibility of penetrable barriers rather than just the impenetrable barriers of previous PGSENMR calculations.     

Lateral phase separations in Escherichia coli membranes

Membranes from unsaturated fatty acid auxotrophs of Escherichia coli were studied by spin labeling and freeze-fracturing. From measurements of the partition of the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) between the aqueous phase and fluid lipids in isolated membranes, temperatures, corresponding to the onset and completion of a lateral phase separation of the membrane phospholipids were determined. By freeze-fracture electron microscopy a change in the distribution of particle in the membrane was observed around the temperature of the onset of the lateral phase separation. When cells were frozen from above that temperature a netlike distribution of particles in the plasma membrane was observed for unfixed preparations. When frozen after fixing with glutaraldehyde the particle distribution was random. In membranes of cells frozen with or without fixing from a temperature below the onset of the phase separation, the particles were aggregated and large areas void of particles were present. This behavior can be understood in terms of the freezing rate with the aid of phase diagrams.     

In Vitro system for the study of bacteriophage phi chi 174 adsorption and eclipse.

An in vitro system was developed for the study of the initial stages of bacteriophage phi chi 174 infection. Escherichia coli C cells were incubated with 20% sucrose and then subjected to cold osmotic shock in 5 mM MgSO4. The concentrated supernatant shock fluid inactivated phi chi 174 with the same kinetics and requirements as for normal infection. Shock fluids prepared from phi chi 174-resistant strains of E. coli did not show this effect. The 114S phage were initially converted into 70S particles, the process termed "eclipse". These structurally altered phages then attached to a component of the shock fluid, producing fast-sedimenting complexes, and eventually released at least a part of their DNA into the medium. The fast-sedimenting complex could be radioactively labeled with oleic acid. Radioactivity was found to co-chromatograph with both biological activity and the majority of the high-molecular-weight carbohydrates present in the shock fluid. It is concluded that E. coli C osmotic shock fluid contains isolated phi chi 174-specific receptor sites composed of lipopolysaccharides. This system conveniently separates the early stages of phage phi chi 174 infection from the intracellular events.     

Pore network modelling of affinity chromatography: determination of the dynamic profiles of the pore diffusivity of beta-galactosidase and its effect on column performance as the loading of beta-galactosidase onto anti-beta-galactosidase varies with time.

A three-dimensional pore network model for diffusion in porous adsorbent particles was employed in a dynamic adsorption model that simulates the adsorption of a solute in porous particles packed in a chromatographic column. The solution of the combined model yielded the dynamic profiles of the pore diffusion coefficient of beta-galactosidase along the radius of porous adsorbent particles and along the length of the column as the loading of beta-galactosidase onto anti-beta-galactosidase immobilized on the surface of the pores of the particles occurred, and, the dynamic adsorptive capacity of the chromatographic column as a function of the design and operational parameters of the chromatographic system. It was found that for a given column length the dynamic profiles of the pore diffusion coefficient were influenced by (a) the superficial fluid velocity in the column, (b) the diameter of the adsorbent particles, and (c) the pore connectivity of the porous structure of the adsorbent particles. The effect of the magnitude of the pore connectivity on the dynamic profiles of the pore diffusion coefficient of beta-galactosidase increased as the diameter of the adsorbent particles and the superficial fluid velocity in the column increased. The dynamic adsorptive capacity of the column increased as (i) the particle diameter and the superficial fluid velocity in the column decreased, and (ii) the column length and the pore connectivity increased. In preparative affinity chromatography, it is desirable to obtain high throughputs within acceptable pressure gradients, and this may require the employment of larger diameter adsorbent particles. In such a case, longer column lengths satisfying acceptable pressure gradients with adsorbent particles having higher pore connectivity values could provide high dynamic adsorptive capacities. An alternative chromatographic system could be comprised of a long column packed with large particles which have fractal pores (fractal particles) that have high pore connectivities and which allow high intraparticle diffusional and convective flow mass transfer rates providing high throughputs and high dynamic adsorptive capacities. If large scale monoliths could be made to be reproducible and operationally stable, they could also offer an alternative mode of operation that could provide high throughputs and high dynamic adsorptive capacities.     

Characterizing the adhesion of motile and nonmotile Escherichia coli to a glass surface using a parallel-plate flow chamber

A parallel-plate flow chamber was used to measure the attachment and detachment rates of Escherichia coli to a glass surface at various fluid velocities. The effect of flagella on adhesion was investigated by performing experiments with several E. coli strains: AW405 (motile); HCB136 (nonmotile mutant with paralyzed flagella); and HCB137 (nonmotile mutant without flagella). We compared the total attachment rates and the fraction of bacteria retained on the surface to determine how the presence and movement of the flagella influence transport to the surface and adhesion strength in this dynamic system. At the lower fluid velocities, there was no significant difference in the total attachment rates for the three bacterial strains; nonmotile strains settled at a rate that was of the same order of magnitude as the diffusion rate of the motile strain. At the highest fluid velocity, the effect of settling was minimized to better illustrate the importance of motility, and the attachment rates of both nonmotile strains were approximately five times slower than that of the motile bacteria. Thus, different processes controlled the attachment rate depending on the parameter regime in which the experiment was performed. The fractions of motile bacteria retained on the glass surface increased with increasing velocity, whereas the opposite trend was found for the nonmotile strains. This suggests that the rotation of the flagella enables cells to detach from the surface (at the lower fluid velocities) and strengthens adhesion (at higher fluid velocities), whereas nonmotile cells detach as a result of shear. There was no significant difference in the initial attachment rates of the two nonmotile species, which suggests that merely the presence of flagella was not important in this stage of biofilm development.     

Cellular spinning in pulsed rotating electric fields

Living cells, dead cells, and other particles suspended in aqueous media of low conductivity can be easily caused to spin if a rotating electrical field is applied. The background and theory are presented for the use of three-electrode systems provided with seriate square electrical pulses to spin cells.     

Influence of the intrinsic membrane protein bacteriorhodopsin on gel-phase domain topology in two-component phase-separated bilayers.

We have investigated the effect of the intrinsic membrane protein bacteriorhodopsin of Halobacterium halobium on the lateral organization of the lipid phase structure in the coexistence region of an equimolar mixture of dimyristoylphos-phatidylcholine and distearoylphosphatidylcholine. The fluorescence recovery after photobleaching (FRAP) technique was used to monitor the diffusion of both a lipid analog (N-(7-nitrobenzoxa-2,3-diazol-4-yl)-dimyristoylphosphatidyle thanolamine, NBD-DMPE) and fluorescein-labeled bacteriorhodopsin (Fl-BR). In the presence of bacteriorhodopsin, the mobile fractions of the two fluorescent probes display a shift of the percolation threshold toward lower temperatures (larger gel-phase fractions), independent of the protein concentration, from 43 degrees C (without bacteriorhodopsin) to 39 degrees C and 41 degrees C for NBD-DMPE and Fl-BR, respectively. Moreover, in the presence of bacteriorhodopsin, the gel-phase domains are much less efficient in restricting the diffusion of both probes than they are in the absence of the protein in the two-phase coexistence region. Bacteriorhodopsin itself, however, obstructs diffusion of NBD-DMPE and Fl-BR to about the same extent in the fluid phase of the two-phase region as it does in the homogeneous fluid phase. These observations suggest that 1) the protein induces the formation of much larger and/or more centrosymmetrical gel-phase domains than those formed in its absence, and 2) bacteriorhodopsin partitions almost equally between the coexisting fluid and gel phases. Although the molecular mechanisms involved are not clear, this phenomenon is fully consistent with the effect of the transmembrane peptide pOmpA of Escherichia coli investigated by electron spin resonance in the same lipid system.     

Improved distance analysis in RNA using network-editing techniques for overcoming errors due to spin diffusion

Multispin magnetization transfer, or spin diffusion, is a significant source of error in NOESY-derived distance measurements for the determination of nucleic acid solution structures. The BD-NOESY and CBD-NOESY experiments, which allow the measurement of interproton distances with greatly reduced contributions from spin diffusion, have been adapted to structural analysis in RNA oligonucleotides. The techniques are applied to a lead-dependent ribozyme (LZ2). We demonstrate the measurement of both aromatic proton–aromatic proton NOEs free of spin diffusion involving the intervening ribose moieties and aromatic proton–ribose proton NOEs free of the efficient cross-relaxation within the ribose ring. In LZ2, the accuracy and precision of the resulting distances are significantly improved. We also find that, by allowing the use of longer mixing times with greater sensitivity, the experimental attenuation of spin diffusion in RNA increases the distance range of interactions that can be analyzed. This effect permits measurement of important long-range distances in LZ2 that are not accessible with standard techniques. Thus, these techniques allow the simultaneous optimization of the number, accuracy, and precision of distance constraints used for RNA structure determinations.     

Individual Escherichia coli cells studied from light scattering with the scanning flow cytometer

BACKGROUND: Flow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells. METHODS: The scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid. RESULTS: The indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth. CONCLUSIONS: The indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied.     

The dispersal of an initial concentration of motile bacteria.

The dispersal of an initial concentration of identical Brownian particles is accurately described by the solution of the conventional diffusion equation, and a diffusion coefficient can be assigned to the assembly of particles. However, the dispersal of an initial concentration of motile bacteria is not well described by the same solution, in spite of the similarity between the random motion of a bacterium and a Brownian particle. Reasons for the failure of the Gaussian solution of the diffusion equation to describe the dispersal of Escherichia coli are discussed. An equation is formulated which gives the concentration of dispersing organisms as a function of space and time if the speed distribution function of the assembly of organism is known and reproduction is suppressed. For three assumed speed distributions the results are compared with concentrations measured by previous authors.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.     

Atomistic simulation studies of cholesteryl oleates: model for the core of lipoprotein particles

We have conducted molecular dynamics simulations to gain insight into the atomic-scale properties of an isotropic system of cholesteryl oleate (CO) molecules. Cholesteryl esters are major constituents of low density lipoprotein particles, the key players in the formation of atherosclerosis, as well as the storage form of cholesterol. Here the aim is to clarify structural and dynamical properties of CO molecules under conditions, which are suggestive of those in the core of low density lipoprotein particles. The simulations in the fluid phase indicate that the system of CO molecules is characterized by an absence of translational order, as expected, while the orientational order between distinct CO molecules is significant at short distances, persisting over a molecular size. As for intramolecular properties, the bonds along the oleate chain are observed to be weakly ordered with respect to the sterol structure, unlike the bonds along the short hydrocarbon chain of cholesterol where the ordering is significant. The orientational distribution of the oleate chain as a whole with respect to the sterol moiety is of broad nature, having a major amount of extended and a less considerable proportion of bended structures. Distinct transient peaks at specific angles also appear. The diffusion of CO molecules is found to be a slow process and characterized by a diffusion coefficient of the order of 2x10(-9) cm2/s. This is considerably slower than diffusion, e.g., in ordered domains of lipid membranes rich in sphingomyelin and cholesterol. Analysis of the rotational diffusion rates and trans-to-gauche transition rates yield results consistent with experiments.     

The distribution of the neural fat body sheath and the accessibility of the extraneural space in the stick insect, Carausius morosus

The fluid compartment delimited at the surface of the central nervous system by the closely applied fat body sheath has been shown to be accessible to molecules of horseradish peroxidase and Indian ink particles. Quantitative analysis indicates that leakage via the intercellular spaces between fat body cells would preclude the possibility of a significant involvement of the fat body cells in sodium regulation of the extraneural fluid. This possibility is further precluded by the absence of a fat body sheath from peripheral nerves.     

Iron oxide particles for molecular magnetic resonance imaging cause transient oxidative stress in rat macrophages

Iron oxide particles are a promising marker in molecular magnetic resonance imaging. They are used to label distinct cell populations either in vitro or in vivo. We investigated for the first time whether small citrate-coated very small superparamagnetic iron oxide particles (VSOPs) can lead to an increase in cellular oxidative stress. We incubated rat macrophages (RAW) in vitro with iron oxide particles. We observed a massive uptake of VSOPs measured both with atomic absorption spectroscopy and with NMR, which could be visualized by confocal laser scanning microscopy. After incubation, cells were lysed and the levels of malonyldialdehyde (MDA) and protein carbonyls were determined. We found a significant increase in both MDA and protein carbonyl levels after incubation with the particles. Surprisingly, 24 h after incubation, a significant indication of oxidative stress could no longer be observed. The increase in oxidative stress seems to be transient and closely linked to the incubation procedure. The iron chelator desferal and the intracellular spin trap PBN caused a significant reduction in oxidative stress to almost control levels. This indicates that the augmentation of oxidative stress is closely linked to the free iron during incubation. Proliferation assays showed that incorporation of VSOPs did not lead to long-term cytotoxic effects even though the iron oxide particles remained in the cell. Magnetic labeling of cells with VSOPs seems to cause transient oxidative conditions not affecting cellular viability and seems to be a usable approach for molecular magnetic resonance imaging.     

Factors restricting diffusion of water-soluble spin labels.

Line broadening of spin label signals is treated in terms of concentration, viscosity, charge and temperature dependencies. Line broadening of spin label signals may be caused either by spin label interactions or by the interaction between a spin label and a second paramagnetic species. Line broadening has been related to collision frequency in the literature and is treated in that way here. Collision frequency is related to diffusion processes in a way that allows information to be obtained about the diffusion environment. Several potential spin label line-broadening agents are compared as to their effectiveness. Small polymer beads with graduated pore sizes are used to show that collisional broadening has a marked dependence on the long-range structure of the diffusion environment. Application of these results to biological diffusion processes is considered.     

Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli

Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.     

Simulation of a Hard-Sphere Fluid in Bicontinuous Random Media

Abstract

The influence of solid-phase connectivity on size-exclusion partitioning and on diffusion of a dilute hard-sphere fluid in overlapping and nonoverlapping spheres models of porous media is investigated using molecular dynamics and Monte Carlo simulation techniques. Four models are examined, two of which are subject to constrained bicontinuity of the pore and solid phases and two in which the solid spheres in the assemblies are randomly distributed in space. It is shown that at moderate to high porosities, connected (bicontinuous) structures lead to a significant increase in the partition and diffusion coefficients when the particles of the pore fluid are of finite size. The consequences of solid phase connectivity are also clearly illustrated in the long-time decay of the velocity autocorrelation function (VACF) of the diffusing particles, particularly in the vicinity of the percolation threshold. Under these conditions the power law exponents on the long-time tail of the VACF are generally found to be higher in connected models than in random systems and the importance of this result is demonstrated using one of the scaling rules of percolation theory. The simulation results are also compared with the predictions of current theories of partitioning and diffusion in random sphere assemblies and, with reference to experimental data available from the literature, it is shown that bicontinuous models are better representations of real porous media.     

Erosion and subsequent transport state of Escherichia coli from cowpats

Processes by which fecal bacteria enter overland flow and their transportation state to surface waters are poorly understood, making the effectiveness of measures designed to intercept this pathway, such as vegetated buffer strips, difficult to predict. Freshly made and aged (up to 30 days) cowpats were exposed to simulated rainfall, and samples of the cowpat material and runoff were collected. Escherichia coli in the runoff samples were separated into attached (to particles) and unattached fractions, and the unattached fraction was analyzed to determine if the cells were clumped. Within cowpats, E. coli grew for 6 to 14 days, rather than following a typical logarithmic die-off curve. E. coli numbers in the runoff correlated with numbers inside the cowpat. Most of the E. coli organisms eroded from the cowpats were transported as single cells, and only a small percentage (about 8%) attached to particles. The erosion of E. coli from cowpats and the state in which the cells were transported did not vary with time within a single rainfall event or over time as the cowpats aged and dried out. These findings indicate that cowpats can remain a significant source of E. coli in overland flow for more than 30 days. As well, most of the E. coli organisms eroded from cowpats will occur as readily transportable single cells.