Microbial attachment to particles in marine and freshwater ecosystems

Scanning electron microscopy observations ofin situ suspended marine and freshwater particles show diverse but similar modes of bacterial and fungal attachment. A survey of Sierra Nevada mountain lakes and pelagic and near-shore waters in the Pacific Ocean indicates that attachment is most noticeable in the near-surface waters where fresh dissolved and particulate input of carbon from phytoplankton and elevated temperatures favor microbial growth. The most common modes of attachment are: adhesive stalk formation, growth on adhesive webs, attachment by the use of pili-like appendages and slimy capsular secretions, and molecular or chemical sorption without the use of visualized structural appendages. Attached microbial growth is accelerated when particulate substrates are supplied, even when they are not rich in organic nutrients. This is the case in the Lake Tahoe basin, where microflora attached to eroded silts can significantly modify the organic carbon and nutrient content of such minerogenous particles.     

Docking of flexible ligands to flexible receptors in solution by molecular dynamics simulation

In this paper, a method of simulating the docking of small flexible ligands to flexible receptors in water is reported. The method is based on molecular dynamics simulations and is an extension of an algorithm previously reported by Di Nola et al. (Di Nola et al., Proteins 1994;19:174-182). The method allows a fast exploration of the receptor surface, using a high temperature of the center of mass translational motion, while the ligand internal motions, the solvent, and the receptor are simulated at room temperature. In addition, the method allows a fast center of mass motion of the ligand, even in solution. The dampening effect of the solvent can be overcome by applying different weights to the interactions between system subsets (solvent, receptor, and ligand). Specific ligand-receptor distances have been used to compare the results of the simulations with the crystal structure. The method is applied, as a test system, to the docking of the phosphocholine to the immunoglobulin McPC603. The results show the similarity of structure between the complex in solution and in the crystal.     

Hepatocyte attachment to laminin is mediated through multiple receptors

The interaction of hepatocytes with the basement membrane glycoprotein laminin was studied using synthetic peptides derived from laminin sequences. Rat hepatocytes bind to laminin and three different sites within the A and B1 chains of laminin were identified. Active laminin peptides include the PA22-2 peptide (close to the carboxyl end of the long arm in the A chain), the RGD-containing peptide, PA21 (in the short arm of the A chain) and the pentapeptide YIGSR (in the short arm of the B1 chain). PA22-2 was the most potent peptide, whereas the other two peptides had somewhat lower activity. Furthermore, hepatocyte attachment to laminin was inhibited by the three peptides, with PA22-2 being the most active. Various proteins from isolated membranes of cell-surface iodinated hepatocytes bound to a laminin affinity column including three immunologically related binding proteins : Mr = 67,000, 45,000, and 32,000. Several proteins--Mr = 80,000, 55,000, and 38,000-36,000--with a lower affinity for laminin were also identified. Affinity chromatography on peptide columns revealed that the PA22-2 peptide specifically bound the Mr = 80,000, 67,000, 45,000, and 32,000 proteins, the PA21 peptide bound the Mr = 45,000 and 38,000-36,000 proteins and the YIGSR peptide column bound the 38,000-36,000 protein. Antisera to a bacterial fusion protein of the 32-kD laminin-binding protein (LBP-32) reacted strongly with the three laminin-binding proteins, Mr = 67,000, 45,000, and 32,000, showing that they are immunologically related. Immunoperoxidase microscopy studies confirmed that these proteins are present within the plasma membrane of the hepatocyte. The antisera inhibited the adhesion of hepatocytes to hepatocytes to laminin by 30%, supporting the finding that these receptors and others mediate the attachment of hepatocytes to several regions of laminin.     

Diffusion-controlled reactions on spherical surfaces. Application to bacteriophage tail fiber attachment.

We have explored the kinetic implications of a model that may account for the acceleration of tail fiber (F) attachment to baseplates (B) by whiskers (W) on bacteriophage T4. The model assumes that a W-F complex is formed initially, and that the tethered fiber then undergoes rotational diffusion until a B-F encounter takes place. In the absence of whiskers, B-F complexes must form unassisted. Formation of a W-F intermediate will accelerate F attachment to B if (a) the bimolecular rate constant for W-F complex formation is larger than that for direct B-F interaction and (b) subsequent rotational diffusion of the tip of F to B is not much slower than the dissociation of W-F. Condition a was investigated by applying a recent theory of orientational effects on translational diffusion-controlled reactions. This theory suggests that substantial rate enhancement is expected if the reaction half-angle theta 0 is larger for W-F than for B-F complex formation. Condition b was investigated by calculating the mean and the variance of the time required for the diffusion of a molecule (the proximal tip of the fiber) on a spherical surface (whose radius is the distance from the tip to the whisker tethering point) into a circular sink (the baseplate site). The mean time is on the order of the inverse rotational diffusion coefficient, DR, of the fiber, but is sensitive to theta 0. Both conditions are satisfied for plausible choices of parameters. The solution to the diffusion equation we have obtained should have application to other physical situations, such as the rate of quenching of a fluorophore as it diffuses on the surface of a spherical membrane into proximity with a quencher.     

A flexible method to align large numbers of biological sequences

Summary A method for the alignment of two or more biological sequences is described. The method is a direct extension of the method of Taylor (1987) incorporating a consensus sequence approach and allows considerable freedom in the control of the clustering of the sequences. At one extreme this is equivalent to the earlier method (Taylor 1987), whereas at the other, the clustering approaches the binary method of Feng and Doolittle (1987). Such freedom allows the program to be adapted to particular problems, which has the important advantage of resulting in considerable savings in computer time, allowing very large problems to be tackled. Besides a detailed analysis of the alignment of the cytochrome c superfamily, the clustering and alignment of the PIR sequence data bank (3500 sequences approx.) is described.     

Active attachment ofAzospirillum brasilense to root surface of non-cereal plants and to sand particles

The rhizosphere bacteriumAzospirillum brasilense Cd adsorbed strongly to light-textured and heavy-textured soils, but only slightly to quartz sand. Bacterial attachment to sand particles was mediated by a network made up of various sizes and shapes of fibrillar material. Inoculation of sand with an aggregate-deficient mutant resulted in no detectable fibrillar formation. Rinsing or agitating the sand, colonized by the wild-type and the mutant, had a greater effect on the mutant than on the parental strain. We propose that bacterial fibrils are essential for anchoring ofA. brasilense to sand. A. brasilense Cd was capable of efficiently colonizing the elongation and root-hair zones of tomato, pepper, cotton and soybean plants as well as of wheat plants. All inoculated plants demonstrated: (i) larger amounts of a mucigel-like substance on the root surface than non-inoculated plants, and (ii) fibrillar material which anchored the bacterial cells to the root surface. These fibrils established also connections between cells within bacterial aggregates. On non-water stressed soybean roots, mostA. brasilense Cd cells occurred as vibroid forms. Whereas, those on roots of water-stressed plants.(wilting) were cyst-like. A lower rhizosphere bacterial population was observed on water-stressed plants. When water stress conditions were eliminated, cells reverted to the vibroid form. A concomitant increase in the bacterial population was observed. We suggest that cyst-like formation is a natural response forA. brasilense Cd in the rhizosphere of water-stressed plants.     

Diffusion-limited forward rate constants in two dimensions. Application to the trapping of cell surface receptors by coated pits.

A variety of receptors are known to aggregate in specialized cell surface structures called coated pits, prior to being internalized when the coated pits close off. At 37 degrees C on human fibroblasts, as well as on other cell types, a recycling process maintains a constant number of coated pits on the cell surface. In this paper, we explore implications for receptor aggregation and internalization of the two types of recycling models that have been proposed for the maintenance of the coated pit concentration. In one model, coated pits alternate between accessible and inaccessible states at fixed locations on the cell surface, while in the other model, coated pits recycle to random locations on the cell surface. We consider receptors that are randomly inserted in the membrane, move by pure diffusion with diffusion coefficient D, and are instantly and irreversibly trapped when they reach a coated pit boundary (the diffusion limit). For such receptors, we calculate for each of the two models: the mean time tau to reach a coated pit, the forward rate constant k+ for the interaction of a receptor with a coated pit, and the fraction phi of receptors aggregated in coated pits. We show that for the parameters that characterize coated pits on human fibroblasts, the way in which coated pits return to the surface has a negligible effect on the values of tau, k+, and phi for mobile receptors, D greater than or equal to 1.0 X 10(-11) cm2/s, but has a substantial effect for "immobile" receptors, D much less than 1 X 10(-11) cm2/s. We present numerical examples to show that it may be possible to distinguish between these models if one can monitor slowly diffusing receptors (D less than 1 X 10(-11) cm2/s) on cells whose coated pits have relatively short lifetimes (less than or equal to 1 min). Finally, we show that for the low-density lipoprotein (LDL) receptor on human fibroblasts (D = 4.5 X 10(-11) cm2/s), the predicted and observed values of K+ and phi are in close agreement. Therefore, even for slowly diffusing LDL receptor, unaided diffusion as the transport mechanism of receptors to coated pits is consistent with measured rates of LDL internalization.     

A simple and flexible device to odorize large stimulation areas

This paper describes a flow dilution olfactometer which allowsthe odorization of large stimulation areas and the easy manipulationof several odorants and/or concentrations. Generation of theodorized air is performed by mixing in two steps the odor vaporcontained in Tedlar bags with a pure air stream flowing continuouslyout of a nozzle. Discrete concentration values are obtainedby using pre-adjusted needle valves to change the vapor flowsampled in the bags. This kind of olfactometer was utilizedto study odor coding in the olfactory bulbs of rats and rabbits.Five Odorants were delivered at concentrations ranging from2 x 10^-4 to 1.5 x 10^-2 of the saturated vapor pressure. Measurementsshowed that lower concentrations can be obtained by fillingthe bags with a more diluted odor vapor. Furthermore, the numberof test odorants can be increased at low cost by increasingthe number of Tedlar bags.     

Droplet sorting of large particles

The systematics of droplet formation conditions for orifices with diameters up to 200 micron are described. Sorting recovery experiments indicate that particles up to 44 micron in diameter can be recovered by charged droplet deflection of two drops with at least 75% recovery. By reducing the jet velocity, a deflection of greater than 1 cm was obtained for all droplet sizes.     

Diffusion coefficients of segmentally flexible macromolecules with two spherical subunits

The translational and rotational diffusion coefficients have been calculated for a simple, segmentally flexible model: the hinged dumbbell (HD). In the HD, two spherical subunits are attached to an universal joint by means of frictionless connectors. In addition to the case in which hydrodynamic interactions are neglected (NI), we have also considered two more cases, including hydrodynamic interaction by means of the Kirkwood-Riseman approximate treatment (KR) and using accurate procedure based in the series expansions for the two-sphere diffusion tensor (SE). Expressions for the friction coefficients of the HD are given for the three cases, and the diffusion coefficients are evaluted inverting the 9 × 9 resistance matrix, for two HDs with different dimensions. The KR treatment, which includes a contribution from the finite volume of the subunits, is shown to be an excellent approximation to the more rigorous procedure. In the NI case for rotation, the various coefficients present different deviations with respect to the SE results. A rough estimate of the errors of the NI relaxation times indicates that they may be smaller than 15% for a HD with identical beads. However, the influence of hydrodynamic interaction should be more important for the rotational diffusivity of a small sphere attached to a larger one. The error of the NI result for the translational diffusion coefficient is of about 25% for the two HDs.     

Ganglioside-dependent cell attachment and endocytosis of murine polyomavirus-like particles

For murine polyomavirus (Py), previous studies suggest the cellular target is a terminal alpha2,3-linked sialic acid. Here, we investigate the binding and uptake of mouse polyomavirus-like particles (PyVLP) derived from bacterially expressed VP1. We find that in fibroblast 3T6 cells, binding of PyVLP was substantially reduced by sialidase treatment, but only moderately affected by protease treatment, suggesting glycolipids such as the sialic acid-containing gangliosides mediate cell attachment. We further tested the entry requirement of PyVLP using the ganglioside-deficient GM95 murine cell line, and find PyVLP binding and entry were reduced in these cells. Finally, we find that addition of gangliosides G(M1), G(D1a), and G(T1b) to GM95 cells restored cellular PyVLP binding and uptake. Taken together, results indicate that gangliosides function in PyVLP cell attachment and endocytosis.     

Asymmetric flows of spherical particles in a cylindrical tube

To study the rheological behavior of blood cells in various flow patterns through narrow vessels, we analyzed numerically the motion of blood cells arranged in one row or two rows in tube flow, at low Reynolds numbers. The particles are assumed to be identical rigid spheres placed periodically along the vessel axis at off-axis positions with equal spacings. The flow field of the suspending fluid in a circular cylindrical tube is analyzed by a finite element method applied to the Stokes equations, and the motion of each particle is simultaneously determined by a force-free and torque-free condition. In both cases of single- and two-file arrangements of the particles, their longitudinal and angular velocities are largely affected by the radial position and the axial spacing between neighboring particles. The apparent viscosity of the asymmetric flows is higher than that of the symmetric flow where particles are located on the tube centerline, and this is more pronounced when particles are placed farther from the tube centerline and when the axial distance between neighboring particles is reduced.     

Conformational flexibility of the N-terminal domain of apolipoprotein a-I bound to spherical lipid particles

Lipid binding of human apolipoprotein A-I (apoA-I) occurs initially through the C-terminal alpha-helices followed by conformational reorganization of the N-terminal helix bundle. This led us to hypothesize that apoA-I has multiple lipid-bound conformations, in which the N-terminal helix bundle adopts either open or closed conformations anchored by the C-terminal domain. To investigate such possible conformations of apoA-I at the surface of a spherical lipid particle, site-specific labeling of the N- and C-terminal helices in apoA-I by N-(1-pyrene)maleimide was employed after substitution of a Cys residue for Val-53 or Phe-229. Neither mutagenesis nor the pyrene labeling caused discernible changes in the lipid-free structure and lipid interaction of apoA-I. Taking advantage of a significant increase in fluorescence when a pyrene-labeled helix is in contact with the lipid surface, we monitored the behaviors of the N- and C-terminal helices upon binding of apoA-I to egg PC small unilamellar vesicles. Comparison of the binding isotherms for pyrene-labeled apoA-I as well as a C-terminal helical peptide suggests that an increase in surface concentration of apoA-I causes dissociation of the N-terminal helix from the surface leaving the C-terminal helix attached. Consistent with this, isothermal titration calorimetry measurements showed that the enthalpy of apoA-I binding to the lipid surface under near saturated conditions is much less exothermic than that for binding at a low surface concentration, indicating the N-terminal helix bundle is out of contact with lipid at high apoA-I surface concentrations. Interestingly, the presence of cholesterol significantly induces the open conformation of the helix bundle. These results provide insight into the multiple lipid-bound conformations that the N-terminal helix bundle of apoA-I can adopt on a lipid or lipoprotein particle, depending upon the availability of space on the surface and the surface composition.     

Full-length prion protein aggregates to amyloid fibrils and spherical particles by distinct pathways

As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full-length recombinant PrP undergoes beta-sheet aggregation on high-pressure-induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, Agg(HP), which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, Agg(HP) transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially beta-sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, I(HP), is formed under high pressure. After pressure release, I(HP) relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, I(HP), constitutes a necessary prerequisite for the formation of amyloids. The use of high-pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids.     

Filamentous bacteriophage contract into hollow spherical particles upon exposure to a chloroform-water interface

The bacteriophage M13 is a 1 μm long filament consisting of a circular single-stranded DNA loop firmly held within a tubular protein capsid. We report here that exposure to a chloroform-water interface initiates a 20 fold contraction of each filament into a hollow protein sphere. In these 0.04 μm diameter particles, termed M13 “spheroids,” two thirds of the DNA is apparently extruded through a hole in the wall of the spheroid; the portion of DNA remaining inside the shell centers about the origins of M13 DNA replication. These results suggest that the filament, upon exposure to a membrane environment, undergoes an ordered change whereby the DNA is released into the cell and the coat protein is changed to a form more easily solubilized by the membrane lipids.     

The flexible attachment of the N-domains to the ClpA ring body allows their use on demand

ClpA is an Hsp100 chaperone that uses the chemical energy of ATP to remodel various protein substrates to prepare them for degradation. It comprises two AAA+ modules and the N-domain, which is attached N-terminally to the first AAA+ module through a linker. On the basis of cryo-electron microscopic and X-ray crystallographic data it has been suggested that the linker confers mobility to the N-domain. In order to define the role of the N-domain in ClpAP-dependent substrate degradation we have generated a ΔN variant at the protein level by introducing a protease cleavage site. The ClpA molecule generated in this way lacks the N-domain and the associated linker but is impaired only slightly in the processing of substrates that are degraded independently of ClpS. In fact, it shows increased catalytic efficiency in the degradation of ssrA-tagged GFP compared to ClpAwt. The role of the linker attaching the N-domain to the bulk of the molecule was probed by characterizing variants with different lengths of the linker. The degradation efficiency of a ClpS-dependent N-end rule substrate, FRliGFP, is reduced for linkers that are shorter or longer than natural linkers but remains the same for the variant where the linker is replaced by an engineered sequence of equivalent length. These results suggest that the flexible attachment of the N-domains to ClpA allows their recruitment to the pore on demand for certain substrates, while allowing them to move out of the way for substrates binding directly to the pore.     

Crowding effects in binary mixtures of rod-like and spherical particles

Crowding effects relevant to the phase stability of binary mixtures of rod-like and spherical particles are investigated by means of Monte Carlo simulations in the isobaric NPT ensemble. The two types of particles are represented, respectively, by freely rotating hard spherocylinders of a moderate aspect ratio (L/sigma = 5) and hard spheres of the same diameter sigma. Molar fractions of spheres ranging xHS = 0.00-0.37 are considered with the aim of characterizing the crowding effects on the liquid crystal phases of the hard spherocylinder fluid induced by the spherical component as depleting agent. We find that the addition of the spherical crowder is beneficial for the stabilization of the layers of the rod-like particles characteristic of the smectic phase. On the contrary, the addition of spheres has a negative impact upon the stability of the nematic phase, where the rod-like particles tend to align collectively parallel to each other. Interestingly, the spheres tend to arrange forming rod-like clusters in the nematic phase and lamellar structures in the smectic phase, which is compensated by the entropy gained by the spherocylinder particles in each phase. The main results are in qualitative agreement with recent experimental and theoretical studies and serve to test the prediction of current equations of state for these types of binary mixtures.     

Obtaining and characterization of spherical particles—new biogenic platforms

The technique of obtaining spherical particles from rod-like plant virus, tobacco mosaic virus, in a preparative scale was developed. The conditions of tobacco mosaic virus isolation for obtaining spherical particles were selected. Spherical particles were examined by methods of electron microscopy, nanoparticle tracking analysis, and dynamic light scattering. Information about inner structure of spherical particles was obtained. High electron density of spherical particles was demonstrated. The analysis of ultrathin sections showed that spherical particles are homogenous within and do not contain any cavities.     

Energy deposition by protons and alpha particles in spherical sites of nanometer to micrometer diameter

Summary Monte Carlo simulated proton- and alpha-particle tracks in water vapor were used to develop an analytical function for calculating number distributions of ionizations induced in spherical sites. For charged particles crossing the site, Fermi-like functions were used to approximate the ionization distributions. Ionization event distributions due to particles passing outside the site were approximated with an exponentially decreasing function. The function parameters were calculated for protons and alpha particles in the energy range 0.3–5.0 MeV/ amu and for site diameters of 1 to 1000 nm. The quality of fit obtained is very good for the particles, energy range and site diameters considered.On leave from the Institute of Nuclear Physics, ul. Radzikowskiego 152, PL-31342 Kraków, Poland     

Modelling of pyrolysis of large wood particles

A fully transient mathematical model has been developed to describe the pyrolysis of large biomass particles. The kinetic model consists of both primary and secondary reactions. The heat transfer model includes conductive and internal convection within the particle and convective and radiative heat transfer between the external surface and the bulk. An implicit Finite Volume Method (FVM) with Tridiagonal Matrix Algorithm (TDMA) is employed to solve the energy conservation equation. Experimental investigations are carried out for wood fines and large wood cylinder and sphere in an electrically heated furnace under inert atmosphere. The model predictions for temperature and mass loss histories are in excellent agreement with experimental results. The effect of internal convection and particle shrinkage on pyrolysis behaviour is investigated and found to be significant. Finally, simulation studies are carried out to analyze the effect of bulk temperature and particle size on total pyrolysis time and the final yield of char.