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MICROBIAL COMMUNITIES AND INTERACTIONS
Membrane Vesicles: an Overlooked Component of the Matrices of Biofilms
Sarah R. Schooling, Terry J. Beveridge
Sarah R. Schooling
Department of Molecular and Cellular Biology and AFMnet-NCE, College of Biological Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada
For correspondence: sschooli@uoguelph.ca
Terry J. Beveridge
DOI: 10.1128/JB.00257-06
The matrix helps define the architecture and infrastructure of biofilms and also contributes to their resilient nature. Although many studies continue to define the properties of both gram-positive and gram-negative bacterial biofilms, there is still much to learn, especially about how structural characteristics help bridge the gap between the chemistry and physical aspects of the matrix. Here, we show that membrane vesicles (MVs), structures derived from the outer membrane of gram-negative bacteria, are a common particulate feature of the matrix of Pseudomonas aeruginosa biofilms. Biofilms grown using different model systems and growth conditions were shown to contain MVs when thin sectioned for transmission electron microscopy, and mechanically disrupted biofilms revealed MVs in association with intercellular material. MVs were also isolated from biofilms by employing techniques for matrix isolation and a modified MV isolation protocol. Together these observations verified the presence and frequency of MVs and indicated that MVs were a definite component of the matrix. Characterization of planktonic and biofilm-derived MVs revealed quantitative and qualitative differences between the two and indicated functional roles, such as proteolytic activity and binding of antibiotics. The ubiquity of MVs was supported by observations of biofilms from a variety of natural environments outside the laboratory and established MVs as common biofilm constituents. MVs appear to be important and relatively unacknowledged particulate components of the matrix of gram-negative or mixed bacterial biofilms.
Biofilms are now commonly perceived as the predominant form in which microorganisms exist in natural and human-made environments. In themselves, the increased persistence and recalcitrance demonstrated by biofilm populations have been a cause for concern in many industrial, health care, and domestic household settings. The exact manner by which biofilm populations become more versatile and resilient than their corresponding planktonic counterparts is currently a matter of great interest but is still not entirely resolved. Yet, in this respect the matrix is an indispensable feature of biofilms. It contributes to the hallmark recalcitrant nature of biofilms by preventing desiccation and phagocytosis, allowing for the accumulation of nutrients, and permitting enhanced resistance to certain inimical agents, as well as influencing fundamental parameters such as rheology and mechanical stability (64, 65). It also helps define the architecture and spatial arrangement of cells (29, 48), and the creation of demarcated niches within the biofilm, i.e., microenvironments. The latter facilitate both phenotypic heterogeneity and species segregation through the creation of environmental continua, e.g., oxygen (16, 67) and pH gradients (12, 59). This adds greater opportunity for diversity and adaptability within a single biofilm. In this manner, the matrix contributes to the intrinsic properties of biofilms and accounts for the great research effort into its underlying chemical and physicochemical properties.
Initial studies of the complexity of the biofilm matrix heavily emphasized exopolysaccharides as the major chemical constituent (14, 15), yet there is now a growing appreciation of other molecules such as proteins, lipids, and nucleic acids (11, 64). These may in turn interact to form higher-order structures, e.g., lattices of intertwined molecules, or particulate structures such as pili (fimbriae), flagella, phage, and membrane vesicles (MVs), all of which have been reported within biofilms (8, 10, 18, 21, 27, 38, 57, 62). Yet, in spite of the tremendous effort to understand the contribution of matrix chemistry to matrix properties, our current knowledge of the ultrastructure of the matrix is still limited (32).
In our current work, we turned our efforts towards the investigation of MVs as promising candidate particulate structures of the matrix of gram-negative and mixed bacterial biofilms. MVs are complex and chemically heterogeneous bilayered structures derived from the outer membrane of a wide variety of gram-negative bacteria, crossing a number of genera (reviewed by references 9 and 43). As MVs bleb from the outer membrane, periplasm fills their lumen and is retained there. The contents of the lumen vary according to the physiology of the parent organism and may consist of a variety of periplasmic constituents such as proteases, alkaline phosphatase, lipases, proelastase, autolysins, and toxins (2, 20, 25, 31, 34, 36, 39). Apart from the lumen contents, another important feature of MVs is their surface chemistry, which is similar to that of the outer membrane of the parent cell although differences in the relative stoichiometry do exist (30, 34). As MVs form, they also retain the intrinsic lipid asymmetry of the outer membrane with most of the lipopolysaccharide (LPS) positioned within the outer leaflet of the membrane (9). LPS has been reported as a component of the matrix (22, 23, 66), possessing possible structural roles (66), and apart from the cells, MVs could represent a major source of LPS within the biofilm matrix. Given the amphipathic nature of LPS, it would likely be arranged in either vesicles or micelles. Since the excision of LPS from the outer membrane with the exclusion of other membrane components (such as proteins or phospholipids) would not readily occur, MVs could be preferred LPS structures in biofilms, especially as these are naturally shed from gram-negative bacteria (9).
In the first part of our work, we used transmission electron microscopy (TEM) of embedded and thin-sectioned biofilms to view the internal structure of the biofilm. Once the presence of MVs was confirmed in thin sections, isolated MVs provided an additional means of proof. In the second part of our investigation we assessed biofilms grown using a number of established model systems and different growth conditions. All of the methods confirmed that MVs were present in all of the biofilms that we studied. Work with a variety of non-laboratory-grown biofilms, taken from natural and human-made environments, also revealed the presence of MVs and established MVs as a common biofilm phenomenon. In the third and final part of this study, we isolated and characterized MVs from planktonic and biofilm populations and found quantitative and qualitative differences between the two as well as indications of roles that MVs may play within biofilms. Collectively, the results suggest that MVs are ubiquitous and important particulate constituents of the biofilm matrix of gram-negative and mixed bacterial biofilms.
Bacterial strain and culture maintenance.Pseudomonas aeruginosa PAO1 was utilized in the study. Cultures were maintained at −20°C in Trypticase soy broth (TSB; BBL, Becton Dickinson and Company) supplemented with glycerol (10%, vol/vol; Fisher) and on Pseudomonas Isolation Agar (Difco; Becton Dickinson and Company) plates and slopes at room temperature. Escherichia coli K-12, Shewanella oneidensis MR-1, and Azotobacter sp. were also used in our study and were maintained on Trypticase soy agar (TSA) plates (BBL, Becton Dickinson and Company) and as frozen stocks.
Biofilm growth.Biofilms were grown using different model growth systems: agar plate (TSA; BBL, Becton Dickinson and Company; 90-mm diameter, Fisher Scientific), flow cell (flat-plate flow cell, 1 mm by 10 mm by 40 mm; Biosurfaces, Inc.), silicone tubing (internal diameter, 1.6 mm) (49), drip reactor (substrata were composed of acetate sheets) (47), and constant-depth film fermentor (CDFF; Mark III; University College Cardiff Consultants Ltd.) (50). All systems were run in a once-flow-through configuration. Flow of medium was allowed to occur for at least 3 h prior to inoculation to precondition the system. Flow was stopped, and inoculation was done through the appropriate port using an early-stationary-phase culture (optical density at 470 nm ≈ 2). Flow was resumed after 1 h. In the case of the drip reactor, substrata were sterilized in 75% (vol/vol) ethanol (2 h), rinsed in sterile water, bathed in culture (1 h), and then aseptically transferred to the assembly. In the case of the CDFF, inoculum was fed into the model for 1 h at 0.1 ml · min−1 and then the flow was switched to sterile medium. With the exception of the agar plate model, unless stated, all of the models used were run at room temperature (∼20°C) with TSB (one-fifth strength) at a flow rate of 0.1 ml · min−1. All biofilms, apart from the agar plate model, were grown for 7 days prior to analyses. Agar plate biofilms were grown on freshly prepared plates at 37°C for 24 h. One milliliter of the inoculum was swirled over the surface, and excess liquid was then removed.
In the second study, a single system was used (the drip reactor) and medium was altered. The media used were TSB (full, 1/5, and 1/10 strength), LB-Miller (Difco; Becton Dickinson and Company; one-fifth strength), and simple salts medium (pH 5.5 and pH 7.5) (44, 54). All experiments were run at room temperature and a flow rate of 0.1 ml · min−1.
Biofilm samples from outside the laboratory.Preformed biofilms were also obtained from sources outside the laboratory. These included samples from domestic household water drains (kitchen and bathroom), rotating biological contactors (municipal wastewater treatment facility), a pulp paper factory, water treatment membranes, the glass walls of a freshwater fish aquarium, a water storage tank, and a riverbed (Grand River, Elora, Ontario, Canada). Samples were excised in situ and transported to the lab for embedding and thin sectioning as described below.
Transmission electron microscopy studies.Samples that were to be assessed by negative staining were prepared as follows. A Formvar- and carbon-coated copper grid (200 mesh; Marivac) was floated, film side down, on 20 μl of sample for 20 s. The grid was removed and the edge gently touched to filter paper (Whatman no. 1; Fisher) so as to wick off excess sample. The grid (sample side) was then washed by being floated on 50 μl of nanopure water, blotted, floated on 10 μl of 2% (wt/vol) uranyl acetate for 10 to 20 s, and blotted dry.
Biofilm samples that were to be thin sectioned were embedded as follows. Each sample was placed in 2.5% glutaraldehyde (vol/vol; in 0.1 M phosphate buffer, pH 7.4; Fisher Scientific) for 0.5 h, rinsed twice with phosphate buffer (0.1 M, 7.4, 5 min), and fixed with osmium tetroxide (1% [wt/vol] in 0.1 M phosphate buffer, pH 7.4; Fisher Scientific) for 0.5 h. Samples were washed twice with nanopure water (5 min) and subjected to an ethanol dehydration series (prepared as volume/volume; Commercial Alcohols Inc.) as follows: 25% (5 min), 50% (5 min), 75% (5 min), 95% (10 min), and 100% (5 min, 5 min, and 10 min). The ethanol was replaced with LR White-ethanol (1:1; London Resin Company, Marivac) for 30 min and then with two changes of 100% LR White over 45 min. The sample was polymerized in fresh LR White (60°C, 1 h) and was ready for thin sectioning. Sections were stained with uranyl acetate and lead citrate.
All samples were examined using a Philips CM10 transmission electron microscope operating at an acceleration voltage of 80 kV under standard operating conditions.
Isolation of matrix and MVs from biofilm.Matrix was isolated from biofilms grown on agar plates (TSA; diameter, 90 mm; one plate per sample). The biofilms were grown to their equivalent early stationary phase (24 h, determined by dry weight), and biomass was scraped from the surface of the plates and resuspended in sterile 0.9% (wt/vol) NaCl. A homogeneous solution was obtained after vortexing (3 min), and this was centrifuged (12,000 × g, 20 min). The supernatant was retained, and the pellet was resuspended in a volume of saline equivalent to that removed and centrifuged. This was repeated three times, and the pooled supernatant was centrifuged at 12,000 × g (20 min) to pellet whole cells. This was repeated three times, the centrifuged supernatant each time being decanted into to a clean tube. The supernatant at this point was essentially isolated matrix material (see Results below), and from this point forward, MV isolation proceeded as for the supernatant obtained from planktonic cultures (51). After ultracentrifugation, the supernatant was retained for further analyses and the MV pellet was resuspended in 50 mM HEPES (pH 6.8) and frozen (−20°C) for later use.
Isolation of MVs from planktonic populations.Planktonic cultures were grown in Erlenmeyer flasks using TSB (one-fifth nominal volume of flask). Inoculation (0.5%, vol/vol) was made from an early-stationary-phase culture, and preparations were incubated at 37°C, 125 rpm. Isolation of planktonic MVs was done according to the protocol of Renelli et al. (51).
Quantitative and qualitative characterization of MVs.Dry weights of samples were obtained by freeze-drying preparations. For dry weight observations, biomass collections were amalgamated in order to obtain sufficient yields.
Diameter values for MVs were determined from micrographs of three independent samples of negatively stained whole mounts, prepared as described previously. Images were archived and analyses were done using the iTEM program (version 5.0; Soft Imaging Systems, Münster, Germany).
CFU were calculated for planktonic and biofilm populations by serial dilution in sterile 0.9% NaCl and triplicate plating of the relevant dilutions on TSA plates. Biofilms were vortexed to obtain a monodisperse suspension before plating. After approximately 20 h of growth, representative plates each having between 30 and 300 colonies were counted and CFU estimates were made. Three independent calculations were performed (n = 9).
3-Deoxy-d-manno-octulosonic acid (Kdo) was evaluated by the periodic acid-thiobarbituric acid method described by Hancock and Poxton (28). Hydrolysis was done for 8 min. Three independent samples were assayed in triplicate (n = 9), and all reagents were purchased from Sigma-Aldrich.
Protein content was quantified using a microbicinchoninic acid protein assay kit (Pierce Bioassay) with bovine serum albumin as the standard. Three independent samples were assayed in triplicate (n = 9).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to assess the protein composition of samples and was done following the method described by Kadurugamuwa and Beveridge (34). Forty micrograms of protein was loaded per lane. Broad-range molecular weight markers were purchased from Bio-Rad. Gels were stained overnight with Coomassie blue R-250 (1 g [wt/vol] in 1 liter of methanol-glacial acetic acid-water [5:1:4]) and destained using several changes of methanol-glacial acetic acid-water (1:2:9) before being washed in water to remove all organic solvents. To ensure repeatability of the final composite gel, SDS-PAGE was performed upon duplicated independent experiments, each of which comprised three separate replicates (n = 6). LPS banding profiles were also assessed (20 μg protein per lane) (34). Zymograms for proteolytic enzyme activity (34) (0.15% [wt/vol] gelatin) were performed using samples prepared with or without reducing agent. Gels were archived using a GS-800 densitometer (Bio-Rad) and the Quantity One program (version 4.4.0; Bio-Rad), with the red filter (Coomassie blue) or green filter (silver stain) in place and at a scanning resolution of 63 μm.
Binding of gentamicin by MVs.Previous studies of gentamicin-induced MV production (34) incubated 8 mg gentamicin/liter culture for 30 min prior to MV harvesting. In order to assess binding of gentamicin specifically to MVs, an aliquot of MVs equivalent in wet weight to that obtained from a planktonic population was incubated with gentamicin (Sigma). This was prepared as a 100-ml MV aliquot equivalent resuspended in 0.2 ml 50 mM HEPES (pH 6.8) to which was added 0.8 mg of gentamicin resuspended in 0.2 ml 50 mM HEPES (pH 6.8). Buffer with no gentamicin served as the negative control. The sample was incubated at room temperature for 30 min, rinsed three times, and concentrated using a Biomax Ultrafree centrifuge filtering system (10-kDa molecular weight cutoff; Millipore). The concentrate was enrobed in an equal volume of Noble agar (4%, wt/vol) and embedded following a less harsh protocol (7) than that outlined earlier so as to minimize alteration of the antigen epitope. Thin sections were immunolabeled (7) using rabbit antigentamicin antiserum (Sigma; 1:100) and goat anti-rabbit immunoglobulin G conjugated to gold (diameter, 10 nm) as the secondary antibody (Sigma; 1:50). Assessment of binding was done using TEM. Less than 1% of bound gold conjugate was seen in the negative control compared to the positive label, and the experiment was repeated in duplicate with observation upon two independent immunolabeled preparations per replicate.
MVs are present within the matrix formed by P. aeruginosa PAO1 biofilms.Preliminary assessment of the physical presence of MVs was done using the simple yet effective agar plate biofilm model. This model has been widely employed (4, 11, 56, 61, 66), primarily due to its ease of use and generous production of biomass and also because it resembles a soft tissue infection (24). As MVs of P. aeruginosa typically have a diameter of 50 to 150 nm (34), our main means of observation was TEM. In this approach, we used TEM of thin-sectioned biofilms to reveal the internal structure of the biofilm (Fig. 1a), and in all sections viewed (n = 8; four sections from two independent preparations), MVs were present within the matrix of the biofilm. The distribution of MVs was variable in that there were areas of little MV accumulation and areas that were dense. While thin sections have a thickness of ∼60 nm and represent a small sampling of the entire specimen, through the observation of a number of samples it was clear that MVs were consistently seen throughout the entire volume of the biofilm. Mechanical disruption of the biofilms and subsequent whole-mount preparations provided an alternative method to show the presence of the MVs. Here, the MVs were seen associated within aggregates of extracellular material (Fig. 2a).
Membrane vesicles in P. aeruginosa PAO1 biofilms. Micrographs of thin sections through embedded P. aeruginosa PAO1 biofilms are shown. The biofilms were grown on a TSA plate (24 h) (a), in a drip reactor (simple salts medium [54], 0.1 ml · min−1; 7 days) (b), in the lumen of silicone tubing (one-fifth-strength TSB, 0.1 ml · min−1; 7 days) (c), and in a flow cell (one-fifth-strength TSB, 0.1 ml · min−1; 7 days) (d). Arrows indicate some of the MVs present in the spaces between the cells, as well as blebbing from the surfaces of cells. Arrows accompanied by the letter F indicate flagella. Bars, 100 nm.
Membrane vesicles were present in mechanically disrupted P. aeruginosa PAO1 biofilm and isolated matrix material and were also isolated following a modified MV isolation protocol. Micrographs of negatively stained whole mounts of P. aeruginosa PAO1 biofilm and isolated components are shown. Biofilms were grown on TSA and mechanically disrupted (a) to observe the components. The matrix was isolated (b), and an MV-enrichment protocol utilized for planktonic MVs was adapted to isolate biofilm MVs from the matrix material (c). Arrows indicate some of the MVs present. Arrows accompanied by the letter P indicate pili or filamentous phage, those with the letter F indicate flagella, and that with the letter A indicates pyocins/aeruginocins. Bars, 100 nm.
MVs are present within biofilms formed under a variety of conditions.The next question that needed to be addressed was whether MVs were a common feature within biofilms or whether our observations were a chance occurrence of the system that we had employed. Biofilms of P. aeruginosa PAO1 were grown using different systems (agar plates, flow cells, drip reactors, tubing, and CDFF) and a variety of media. The different systems were chosen to represent some of the conditions under which natural biofilms are formed: saturated (flow cell and silicone tubing), unsaturated (agar plate and drip reactor), aggressive shear (CDFF), fluid flow (drip reactor, silicone tubing, and flow cell), and closed (agar plate) environments. Figure 1 shows micrographs of thin sections through these biofilms (Fig. 1a to d).
Again, in contrast with MVs from planktonic bacteria that migrate from the parent cell once liberated, biofilm MVs were found to accrete within the extracellular matrix. This was a consistent feature of all of the different biofilm systems and growth conditions assessed and supported our previous observations. MVs were also located at the substratum-bacterium interface (Fig. 1b), as a result of either predeposition before cell attachment or production after cell adhesion. In addition, negative stains of effluent from each growth system revealed that MVs were also shed (data not shown). That is, the biofilms not only maintained substantial numbers of MVs within their matrices but also dispersed MVs into the external environment. In this manner, biofilms could effectively serve as both "safe house" factories and depositories of MVs, as well as export depots during processes such as infection or predation and ecological succession. This bears a number of implications in that the traditional boundaries of both cell and intact biofilm become blurred. MVs, as extracellular extensions of cells, may then manifest their effect(s) not only throughout the matrix but also beyond the borders of the biofilm.
Finally, biofilms of Shewanella oneidensis MR-1, Escherichia coli K-12, and an Azotobacter sp., all grown using a drip reactor and one-fifth-strength TSB, were prepared for viewing as thin sections (data not shown). Again, MVs were found to be part of the matrix of these gram-negative bacterial biofilms. This was in agreement with the concept that MV production is a normal function of the physiology of gram-negative bacteria and, in our study, that MVs are a component of the matrix of gram-negative bacterial biofilms.
MVs are found within isolated biofilm matrix material and can be enriched from this fraction.Biofilms were suspended in saline, and the cells were separated from the matrix by differential centrifugation. Examination of the cell pellet by TEM showed that the majority of the material had been disassociated from the cells, yet ca. 10 to 15% remained bound to cells in the pellet. We did not employ severe chemical or mechanical treatment to fully strip this material from the cells due to concerns about possible cell damage or lysis and subsequent sample contamination. For example, EDTA, a known perturbant of the outer membrane that artificially generates MVs (T. J. Beveridge, unpublished data), has sometimes been employed to increase the yield of isolated extracellular polymeric material (which would be contaminated with abnormal quantities of MVs). TEM of negative stains of our extracted matrix showed the presence of MVs, as well as other particulate structures such as flagella, pyocins, and pili/filamentous phage (10) (Fig. 2b). Since MVs are blebs from the outer membrane, then Kdo, a marker for LPS, should be present. Indeed, our results were in agreement with this (Table 2). Previous reports have indicated Kdo to be present within matrix material (22, 23, 66), and we believe that this "matrix LPS" is partly due to the presence of MVs (see below).
The matrix-associated MVs were then isolated as an enriched preparation by integrating an MV isolation protocol based upon that of Renelli et al. (51). After centrifugation at 150,000 × g, the pellet was enriched with MVs; however, other particulates were also still present—flagella, pili, and pyocins (Fig. 2c). This was consistent with similar observations of planktonic pellets, since a low-speed centrifugation of the resuspended pellet largely reduced the presence of such contaminating materials from this preparation (51). Kdo values (Table 2) indicated that this enriched pellet contained a substantial 52% of the LPS present within the matrix material, and dry weight values indicated that this pellet represented 18% (wt/wt) of the matrix material (Table 1). TEM observations of negative stains of the associated supernatant indicated no MVs to be present, and it is possible that LPS may be associated with other molecules such as polysaccharides, nucleic acids, and proteins which, under the present conditions, did not pellet out. However, MVs clearly represent a major reservoir of LPS within the matrix.
Dry weights of biofilm and planktonic fractions
MVs from planktonic and biofilm populations differ both quantitatively and qualitatively.Upon isolation of the MV-enriched pellets, it was surprising that the volume, color, and consistency of these biofilm MV-enriched pellets contrasted sharply with those obtained from planktonic populations (Fig. 3). The biofilm pellets were consistently larger than the planktonic ones even though they were derived from parent populations of approximately equivalent dry weight and CFU (Table 1 and legend to Fig. 3), and were of a mid- to dark brown color whereas the planktonic pellets were small and black. The biofilm pellet was more gelatinous than that from planktonic cells (more friable). Negative stains revealed that both preparations had MVs as a major pellet constituent. Together, these observations suggested both qualitative and quantitative differences that required further investigation.
MV-enriched ultracentrifuged pellet from planktonic and biofilm populations. MV-enriched ultracentrifuged pellets obtained from CFU-equivalent biofilm populations (right) were of a higher volume and a different color and consistency (more gelatinous) than those from corresponding planktonic populations (left). This visual difference was supported by values for dry weights and total protein and Kdo contents.
The low-speed centrifugation used to clean contaminating materials from isolated planktonic MVs (51) was implemented for both planktonic and biofilm MVs. In the case of the former, there was an effective reduction in the presence of flagella, pyocins, and pili as determined by TEM of negatively stained samples (Fig. 4a). Biofilm MVs displayed an anomalous behavior: MVs did sediment, but the vast majority remained in suspension and could be harvested from the supernatant (TEM) (Fig. 4c). This behavior may be the result of either buoyant density differences or their smaller size (Fig. 4a and 4c; see below). Furthermore, the pellet consisted of an entanglement of flagella, pili, pyocins, and some MVs (Fig. 4b). The MVs that did pellet tended to have a larger diameter and were more similar in size to the planktonic MVs. Kdo values of the two fractions supported this observation, with more Kdo present in the supernatant than in the pellet (Table 2). In our hands, this sedimentation behavior was a consistent feature only if the ultracentrifuged sample was sufficiently diluted and resuspended to monodispersity; overloading of the sample resulted in loss of separation. From this point forward, unless otherwise indicated, work with planktonic MVs refers to MV isolations that were pelleted and work with biofilm MVs refers to MVs that remained in suspension. In both instances there was loss of sample in that MVs did not totally migrate to one or the other of the collected fractions with either population.
Outcome of low-speed centrifugation on biofilm MVs. MV-enriched ultracentrifuged pellets were resuspended in HEPES buffer and centrifuged at 16,000 × g for 30 min. Planktonic-derived MVs pelleted out (a), whereas the pellet obtained from a biofilm preparation consisted mainly of flagella, pili, and some MVs (b). The vast majority of the MVs remained in the supernatant (c). Bars, 200 nm.
Kdo values in the various fractions isolated from biofilms
MV yield comparisons were made using the main parameters of dry weight and Kdo. In all instances, under the stated growth conditions, dry weight values indicated that the biofilms exceeded planktonic productivity (Table 1). Furthermore, given that the biofilm MV wash fraction contained the majority of MVs (see previous paragraph), a revised comparison of the normalized dry weights of the MV-rich fraction from the two populations yields a planktonic/biofilm ratio of 16.38. Kdo values (Table 2) also indicated a higher yield for biofilm populations, and the ratio of Kdo values (normalized to 1 × 109 CFU) indicated that biofilms produced ca. 120 times more MV-associated Kdo (242 ng versus 2 ng). This sevenfold discrepancy between the weight and Kdo ratios (16.4 versus 121) suggested that biofilm MVs contained more Kdo per unit weight than did their planktonic counterparts. Kdo/protein ratios for the planktonic and biofilm MVs also reflected a similar trend. Planktonic MVs contained 10,650 ng Kdo/g culture protein with a corresponding 15.59 μg Kdo/mg MV protein, whereas biofilm MVs possessed 75,423 ng Kdo/g culture protein with 109.63 μg Kdo/mg MV protein. In both instances, the difference between the respective ratios was sevenfold. SDS-PAGE of Hitchcock-Brown preparations also supported this observation (Fig. 5a), and biofilm MVs stained more intensely than planktonic MVs when equivalent amounts of protein per sample were loaded. While this sevenfold difference initially appeared odd, a possible explanation may be found within the size distribution data for planktonic and biofilm MV populations (Table 3). Biofilm MVs were generally smaller, as observed by negative stains and thin sections, which may in part also explain the sedimentation differences noted above. The average MV diameter values obtained for biofilm and planktonic populations were 45 and 86 nm, with corresponding medians of 40 and 85 nm, respectively. This smaller diameter meant that biofilm MVs have an average surface area-to-volume ratio that is approximately twice that of planktonic MVs. This would be reflected in the higher Kdo ratio, but it does not entirely account for the discrepancy in values and supports a genuine difference between the two populations. Our planktonic Kdo values are similar to previously published values (i.e., 66 nmol Kdo/mg protein compared to 41 nmol Kdo/mg protein) (51), yet the values obtained for the biofilm MVs had an astonishing 460 nmol Kdo/mg protein. One possible explanation is that this was a deliberate alteration of the Kdo/protein ratio by the biofilm cells or that the outer membranes from which the MVs are derived are different in planktonic and biofilm populations. Either possibility bears a number of interesting implications. For example, one may consider the reactivity of the LPS molecule and the interactions that it may mediate, the possibility of less protein being packaged into the MV lumen, or the chance that relatively fewer outer membrane proteins occur. The latter may minimize the chances of detection by the host immune system.
SDS-PAGE analyses of MVs. SDS-PAGE was used to analyze both planktonic and biofilm MVs. The relative amounts of LPS and protein were compared using silver staining (a), and protein profiles were compared using Coomassie blue staining (b). Finally, proteolytic activity was examined using zymogram assays with 0.15% (wt/vol) gelatin as the substrate, in the absence and presence of a reducing agent (c). Arrows indicate zones of proteolysis. Twenty micrograms of MV protein was used per well in panel a, and 40 μg of MV protein was used per well in panels b and c.
MV diameter distributionsa
Indeed, SDS-PAGE analyses of the two MV-rich populations demonstrated that apart from the quantitative yield differences earlier described, there were dissimilarities other than size and centrifugal sedimentation properties (Fig. 5b). The planktonic and biofilm MV profiles demonstrated that, although there were proteins in common, there were also a few prominent differences. For example, biofilm MVs have a denser band at around the 55-kDa mark, which could be alkaline protease (see below). In general, however, biofilm MVs had less diversity in the bands present than those from planktonic cells, again supporting the notion that a more limited selection of proteins is being secreted as part of the biofilm MV complement. Future studies into the nature of the different proteins would shed light onto this and perhaps reveal some commonality in those proteins that are present.
Investigations into the functionality of biofilm MVs.Due to the large economic expense that MV production entails to the cell, it has long been argued (and demonstrated) that they are not nonsensically produced. Among the diverse functions ascribed to planktonic MVs are their roles as a novel secretory pathway (37, 60), the delivery of virulence factors (20, 30, 31, 34), cell-to-cell signals (42), cell aggregation (19, 25, 35), metal immobilization and redox processes affecting minerals, inactivation of antimicrobials by enzymatic degradation (e.g., β-lactamases) (13) or by binding (26), the selection and destruction of non-self cells (40), immune-modulating substances (1, 45, 46), and (somewhat controversially) the transfer of genetic material (17, 39, 68). All of these processes do occur within the scope of a biofilm, and there is no apparent reason why MVs should not participate in them. Furthermore, given that the MVs are shed, these processes may be relayed to actively have an effect outside of the confines of the biofilm, even at a good distance from it.
Planktonic MVs have been reported to contain potent hydrolytic enzymes such as proteases and peptidoglycan hydrolases (34, 40). As the SDS-PAGE analyses (Fig. 5b) had suggested the possibility of a protease, we performed zymogram assays (34). Samples were prepared both in the presence and in the absence of a reducing agent. This was essential as certain enzymes lose their functionality or give an enhanced response in the presence of a reducing agent (41). Proteolytic activity was found associated with both the planktonic and biofilm MVs (Fig. 5c), under both conditions. However, in both instances using the same sample concentrations, biofilm MVs demonstrated greater proteolytic activity, as denoted by the larger zones of clearing. Under nonreducing conditions, four bands were seen: a very diffuse band at the 20-kDa region, a band at approximately 55 kDa, and two bands running at 80 to 90 kDa. It was noted that the Rf values of some bands differed from those run on a gel without gelatin. Under reducing conditions, only one prominent band was present at approximately 55 kDa. This band is consistent with alkaline protease, as is its behavior under reducing conditions (41). It is possible that the diffuse 20-kDa-region band seen under nonreducing conditions was PrpL (27 kDa) (53). However, the evidence remains that biofilm MVs contained more proteolytic activity than those from planktonic cells, an important consideration when thinking of the roles that MVs may play in pathogenesis, release of nutrients, and perhaps surface modification. Furthermore, there are reports of proteases within the matrices of biofilms (53), and clearly, a proportion of these reside in MVs.
In the second study, we assessed the ability of MVs to bind antibiotics, in particular gentamicin. The binding of this cationic aminoglycoside is well studied (33) and a logical starting point. Both planktonic and biofilm MVs were incubated with gentamicin, and binding was assessed by immunolabeling and TEM (Fig. 6). The micrographs clearly demonstrate that biofilm MVs did bind gentamicin, and the label revealed the aminoglycoside to be located at the outer and inner faces of the membrane, as well as within the lumen. Planktonic MVs were also shown to interact with gentamicin (data not shown). Clearly, since gentamicin was attached to all regions of the MVs, the antibiotic must have penetrated entirely through the MV, indicating strong or even disruptive bilayer interaction. This is not surprising since highly cationic substances, such as aminoglycoside antibiotics, can displace essential metal cations and disrupt lipid-packing order (33). Not all MVs showed labeling, but this is expected and was seen in other studies (34). This could be due to a number of factors, e.g., diminished antibody-antigen affinity due either to the antibody itself (the manufacturer's sheet states that binding is up to 40% of gentamicin present for a radioimmunoassay) or antigen alteration during the embedding protocol, or it could be that the bound gentamicin was simply not accessible in the thin section. Indeed, in our experience the consistent labeling of these small vesicles was remarkably efficient considering their size and likely exposure in thin section (7). Yet, it remains that the MVs did bind exogenous gentamicin, and given the surface chemistry of MVs, the vesicles could also bind other extraneous compounds. As a general property of biofilm MVs, they could act as decoys or "sponges" to reduce inimical agents within biofilms before they affect cells. Since MVs are released from biofilms, the concentration of such agents within the biofilm would be reduced and MVs dispersed from a biofilm could serve to bind agent prior to contact with the biofilm. This could be another way in which biofilms are protected from antimicrobials. If so, it represents a simple but effective mechanism and a route that we hope to explore in greater detail.
Binding of gentamicin by MVs. Shown are micrographs of biofilm MVs that had been incubated with gentamicin prior to embedding and thin sectioning, probed with a primary antibody specific for gentamicin, and developed with a secondary gold-conjugated antibody. Negative controls (unreacted MVs) indicated good specificity. The location of immunolabel indicated where gentamicin interacted with both the outer and the inner leaflets of the membranes (a, b, c, d, f, and g) and could also be found within the lumen of MVs (b, c, e, and f). Note the variety of MV sizes and how, in some instances, the size approached the diameter of the label (10 nm; panel d, indicated by arrow). Bars, 20 nm.
MVs are also found within naturally occurring biofilms.Laboratory-grown biofilms are very different from natural biofilms. The former are often monocultures subjected to a constant and reasonable flow of nutrients, whereas the latter are a complex mixture of cultures that are subjected to a variable and often harsh set of environmental conditions. It was therefore necessary to sample natural biofilms to ensure that MVs were a natural trait of these systems. A number of different environmental conditions were sampled. These included domestic water drains, sewage and water treatment plants, pulp and paper manufacturers, freshwater fish aquariums, water storage tanks, and riverbeds. MVs were seen in all biofilms where gram-negative bacteria were found. There was a more discontinuous distribution of MVs than that seen in any of the PAO1 biofilms. This was presumably related to the distribution of gram-negative bacteria present since MVs were shed and found close to these regions (Fig. 7), although other factors such as environmental conditions could play a role (30, 36, 52). Additionally, environmental biofilms often incorporated more extraneous material (e.g., large plant fibers, mineral grains, large protists, etc.), effectively adding to the bulk of the biofilm. Our laboratory has extensive experience in sampling microbiota growing as biofilms or flocs (especially those of marine "whiting" events) from a number of dissimilar natural environments. We have never encountered a natural setting where gram-negative bacteria were present that did not contain MVs.
FIG.7.
Membrane vesicles in biofilms from outside the lab. Shown are micrographs of thin sections through conventionally embedded biofilms obtained from a domestic bathroom drain (a) and a water treatment membrane (b). Note the presence of different cell types. Arrows indicate some of the MVs present in the spaces between the cells, as well as blebbing from the surfaces of cells (panel b, white arrows). Bars, 1,000 nm.
Potential of biofilm MVs.The overall goal of our study was to establish MVs as a bona fide component of the matrix of gram-negative bacterial biofilms. Through our TEM studies, we found that MVs were a definite and consistent component of the matrix of gram-negative bacterial biofilms. Furthermore, they were shed from the biofilms, disseminating into the external environment where they could serve as a source of extracellular activity both in and ex biofilm. Under defined conditions, we found that these exquisite structures could account for up to 16.8% (wt/wt) of the matrix material and more than 50% of the Kdo found within the matrix material. The presence of these LPS-bearing structures firmly places LPS as a matrix component and not a contaminant or procedural artifact. Additionally, while Kdo values have been used to show that LPS is a minor-percentage matrix component, Kdo represents only 2 to 6% of the LPS (wt/wt) of P. aeruginosa PAO1, a fact that is often overlooked. These biofilm MVs were also found to substantially differ from their planktonic counterparts in terms of their physical dimensions and properties, as well as in their chemistry. It was impossible for us to probe MVs for all potential functions, but we have certainly indicated a few—the packaging of virulence factors such as proteases and the binding of antibiotics.
In some of the samples, MVs were located at the substratum-biofilm interface and either were deposited prior to biofilm development or were produced by the biofilm, i.e., cells can alter the surface properties of the substratum either pre- or post-biofilm formation. Planktonic MVs derived from Bacteroides gingivalis adhere to hydroxyapatite and facilitate the attachment of Streptococcus sanguis (55). Other studies have indicated that MVs can also mediate aggregation of cells (19, 35, 43). Can cells then use MVs to influence early-stage biofilm processes such as adhesion? It is important to recognize that biofilms form on an enormous variety of substrata that range from inert mineral faces to high-carbon films (e.g., cellulose and chitin) to soft tissue surfaces (plant, mammalian, etc.); MVs could be actively or passively altering substratum surface properties. Alternatively, the MVs could simply be entrapped between the substratum and the producing cells.
Substratum-associated MVs, however, account for only a fraction of the total. In considering the size, frequency, and chemical nature of MVs, these occupied a substantial volume of the biofilm. It is also important to remember that MVs are characteristic of the producing cell and its phenotype, possessing serotype-specific LPS and outer membrane proteins, which provide a unique surface chemistry for environmental interactions. Available surface molecules or intrinsic reducing activity could affect and be affected by the redox and pH of the local microenvironment surrounding groups of cells within the biofilm. These very same interactions might play a role in stabilizing polymers, ions, and other components of the matrix, all contributing to the properties of a microenvironment. Close to the producer cells, through small-scale interactions, they could have an impact by providing sorbative or inactivating power on extraneous agents, thereby protecting cells. Additionally, MVs could also be capable of interacting with polymers within the biofilm, e.g., DNA and polysaccharide, influencing their ability for entanglement. This must alter the intrinsic properties of the polymers through electrostatic or hydrophobic interaction and binding. Indeed, DNA, a substantial ingredient of the matrix of P. aeruginosa biofilms (3, 63), is a proven constituent of MVs and can be associated with the lumen or the membrane surface (34, 51). Accordingly, some of this matrix DNA could actually be MV DNA. There could also be strong associations between exogenous matrix DNA and MVs as well as other biofilm particulates (58). It is even possible that MVs could contain enzymes capable of altering polymers (e.g., nucleases, polysaccharases, and epimerases). All particulates and their interactions with the matrix must have a strong impact on the rheological properties of biofilms.
Apart from the possibility of physical interaction with extracellular polymers, MVs have been shown to contain active periplasmic components, e.g., enzymes and toxins. For example, certain strains of P. aeruginosa produce and package β-lactamase into MVs, which can then degrade β-lactam antibiotics (13). It is interesting that biofilms exposed to β-lactams have an increased synthesis of β-lactamase (5, 6). In addition, Porphyromonas gingivalis produces MVs that bind and sequester chlorhexidine (26). It is then possible that biofilm cells can be induced to shed MVs with active properties that would neutralize inimical agents designed to attack and destroy biofilms.
Finally, biofilm MVs appear to be independent, extracellular extensions of the cell, which broaden the traditional boundaries of the cell. These boundaries include regions within the biofilm itself and beyond its confines. In the latter context, we could imagine a biofilm being a location from which MVs are liberated to manifest certain properties in the broad-scale environment. These could be intrinsic chemical properties (e.g., those affecting geochemical conditions within a geological horizon) or active components (e.g., virulence factors) to promote a desired bioeffect. Biofilms would be much more long-lasting durable depots for these bioactive MV particles than planktonic cells and would be a continual source of such environmentally altering substances. Since many of the active ingredients are encapsulated within the MVs, they would be better protected from antagonistic external factors. Furthermore, since MVs mimic the bacterial cell surface, they would have strong adhesive properties or (even) specific adhesions for attachment (e.g., on certain tissue types).
In summary, there is an increased awareness of biofilms and their abundance in nature. Hardly a month goes by without new reports on the properties of biofilms, yet few have concentrated on the particulate properties of the biofilm matrix. While our nascent understanding of the matrices of biofilms led to the belief that these consisted primarily of exopolysaccharides, this study, along with many others, consolidates this as an erroneous perception. The matrix is a complex amalgam, comprised of polymers and macromolecules, as well as particulate structures such as discarded pili and flagella. Here, in our report, we emphasize MVs as being among the most important because of their intrinsic surface properties and active constituents. We hope this will encourage further studies of these fascinating nanoparticles.
This work was made possible by an NSERC-Discovery grant to T.J.B. for general operating funds and by CBDN-NCE and AFMnet-NCE grants to T.J.B., which helped cover salaries. The TEM was performed in the NSERC Guelph Regional Integrated Imaging Facility (GRIIF), which is partially funded by an NSERC-MFA grant to T.J.B.
We thank Corinne Michaud for preliminary work that led to the development of this study, Dianne Moyles and Robert Harris for expert guidance with the TEM-related aspects of this study, and Anuradha Saxena for technical assistance.
Received 18 February 2006.
Accepted 8 June 2006.
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Journal of Bacteriology Aug 2006, 188 (16) 5945-5957; DOI: 10.1128/JB.00257-06
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2009 has been a good year for operating systems, or at least the past few months have been. Mac's latest operating system, Snow Leopard was released just a couple of months ago and this week two of the best operating system ever devised are released: Windows 7 and Ubuntu's Karmic Koala.
Microsoft's new operating system has a lot to make up for, namely Vista and the lack of a decent Microsoft operating system for almost a decade.
The fact that the most used Microsoft operating system is still Windows XP, which was launched eight years ago, demonstrates just how tough a job selling Windows 7 is going to be for Microsoft, especially after the Vista disaster.
So what's new in Windows 7? Very little compared to Vista, indeed some critics are calling Windows 7, a Vista service pack or update. Certainly Windows 7 is the operating system that Vista should have been and there are some great improvements.
This is the important improvement for most people. Vista had a tendency to turn their fast PC into a slow coach, making the user experience a frustration for most people. These issues have been fixed in Windows 7 and the operating system is very nippy, even on netbooks. Yes, Windows 7 even works on netbooks, something that Vista could never do.
Windows 7 boots swiftly, the desktop effects work brilliantly, even on a low powered netbook, and the suspend/resume is almost instant. Performance wise, Windows 7 is a joy to use, it is brisk, even with many windows open and even on our netbook we never noticed any lag.
In Windows 7 Microsoft have altered the taskbar, no longer does it display the number of open windows, instead it just displays a single icon for each application, which, when hovered over, displays thumbnails of the open windows. You can also 'pin' applications to the taskbar, similar to the quick launch area of XP and Vista, and they glow a different colour when they have an application window open.
Initially the new taskbar seemed like a great idea, but this quickly becomes annoying when you have two or more firefox or explorer windows open and have to hover over the icon, and then the thumbnail, and then click it the thumbnail just to get the window you wanted; only to have it revert back to the previous window when you didn't click it hard enough or in the right place!
Windows 7 has gadgets similar to Vista, except they are no longer in a sidebar by default, instead floating on the desktop in a similar way to those on previous versions of Ubuntu.
Windows 7 now also supports themes, something that has been promised since XP. You can now alter your theme and download new themes. There are also country specific themes included, in the UK theme you get desktop backgrounds of places like Stonehenge, Tower Bridge and the White Cliffs of Dover.
Another great feature is the slideshow, which changes your desktop background automatically after a certain period. This is another feature that should have been available years ago, and it is great to be able to choose your own pictures and have the desktop change periodically. Previously third party applications were needed to do this, indeed they still are in Ubuntu.
Although introduced in Vista, the program search in the start menu is also worth a mention. After using XP for many years this is a great time saver, simply start typing the name of a program and Windows begins narrowing it down to a few possibilities and then click the program you require. So simple and yet so useful.
Libraries are one of the changes that I found annoying at first, but have since found to be quite useful. Windows 7 automatically sorts your files into categories (or Libraries) of Music, Videos, Pictures and Documents. This makes it much easier to find files, however not so easy when sharing files as it can cause confusion as to actual file locations.
As usual Windows 7 comes in several varieties, the cheapest home retail version available in the UK is Windows 7 Home Premium. Don't be fooled by the 'Premium' in the title, this is the worst one available, yet it will still set you back about £100. If you want useful things such as the very good backup utility, drive encryption, remote desktop and Windows XP mode (to run your old XP programs), well that costs extra, about £50 extra!
To get all the features of Windows 7, one needs to stump up about £180.
Having used Ubuntu for many years, initially being drawn to it just as something different (it was a long wait from XP to Vista), it has to be said that many of their recent operating systems have been more than a little underwhelming. Indeed the last few appear to have been a step backwards in some cases.
Past disappointment and releasing their new OS at the same time as the top dog, meant that Ubuntu also had something to prove.
Performance in the last couple of Ubuntu operating systems has also been less than impressive, Intrepid Ibex 8.10 and Jaunty Jackalope 9.04 particularly, displayed degraded performance on laptops that had worked great under Hardy Heron 8.04 and Gutsy Gibbon 7.10.
Karmic Koala 9.10 however is the fastest Ubuntu has been for a while. Boot time is very fast, boot time on this laptop was just under a minute on Gutsy Gibbon 7.10, but it hasn't been near that since. Running 9.10, this laptop boots in around 30 seconds. Suspend/Resume is also much quicker, indeed resume is almost instant, faster even than Windows 7. Shutdown is also super fast.
The desktop effects that worked flawlessly on even low powered laptops in the Hardy Heron 8.04 days, have suffered from inexplicable CPU spikes and lags since, but now seem to be on top form again.
Karmic Koala has made Ubuntu swift again, very similar to Windows 7, but even so it has to be said that Windows 7 is still smoother; the long speed advantage that Ubuntu has had over Windows (and that always amazed Windows users) may finally have ended with Windows 7.
As usual Ubuntu is far more customisable than Windows, with more gadgets and far more themes than Windows 7 (Ubuntu even has Windows 7 style themes). However it is a shame that Ubuntu's gadgets, or screenlets, are not installed by default, but at least like everything else on Ubuntu, it is easily installed.
Like other versions of Ubuntu, 9.10 offers the ability to change the desktop effects, but again this isn't installed by default, but once installed the user can choose from many, many different effects for closing, opening or minimising windows.
Sadly there is no option for timed desktop background changes like on Windows 7, at least without installing additional software.
Arguably the best piece of software on Ubuntu is Gnome-Do, this is like a cross between Windows program search and Google Desktop Search, but better than both. With a simple keyboard combination (Super/Windows Key + Spacebar) a box pops up that can do everything from simply finding a program as you type its name to searching for files, definition of words or even posting to Twitter.
Once again this isn't installed by default but is available to install. Installation is pretty simple however, Ubuntu have replaced the Add/Remove program with Ubuntu Software Store which allows the user to search for software and install it with a single click. Future versions will also allow developers to sell their software via the store but at present only free software is available.
This makes things even easier than before, and with the added bonus of an Installed programs list, it means that Ubuntu users can finally keep track of what is installed and uninstall all those programs that were installed, used once and promptly forgotten all about. A great move by Ubuntu, but something that has been possible in Windows since its earliest days.
Obviously Ubuntu is free, but to give a basis for comparison it comes in only one variety - Ultimate. Ubuntu Karmic Koala offers encryption options during start up, such as creating an encrypted home partition, Remote Desktop software by default, and simple backup and Windows XP emulators can easily be installed, all for free.
Karmic Koala also offers 2GB of cloud storage with Ubuntu One, again installed by default and also free.
Ubuntu's new operating system is its best yet, it is fast, slick, and seems to get Ubuntu back on track, however other than in performance, it differs little from other versions of Ubuntu.
Windows 7 basically is an updated Vista, but it is also arguably Windows best ever operating system. Both Windows 7 and Ubuntu 9.10 offer the best performance and stability yet seen from their respective series, but Windows 7 seems to edge it in terms of speed and stability, having never crashed on our netbook.
The let downs in Windows 7 are the lack of ability to alter or customise the Aero settings, which, although they look great, quickly become boring, and cannot compete with Ubuntu effects like Burn, Paper Airplane or Beam; and the fact that the Home version is lacking some vital components, particularly XP mode, which you'd expect in a £100 OS.
Apple were able to offer their great operating system Snow Leopard for just £29.99, yet Microsoft, who control 90% of the PC market, charge £100 for their bog standard version.
This is a great offering from Ubuntu, but it also has to be said that Windows 7 is also a great operating system and shows that Microsoft have really raised their game, and we have the likes of Ubuntu and Apple to thank for that. However Windows 7 doesn't feel like a complete operating system, and not just because of the things missing from the home version.
Ubuntu 9.10 too feels like it is still unfinished, and doesn't quite feel as polished as Windows 7.
Overall I'd say that Windows 7 just edges it as the best operating system around, but when you take into account the cost to upgrade, the fact that it doesn't include any Office or other useful software for free, Ubuntu 9.10 seems the better option.
Vista has 20% of the desktop market, XP about 70%, so Microsoft is going to have its work cut out tempting those users to upgrade, especially with such steep pricing. These are tough economic times, so forking out around £200 on a new OS and assorted software, or more on a new computer with Windows 7, is likely to be viewed as an unnecessary expense, particularly if their current computer is running fine.
Add to that Ubuntu's excellent Karmic Koala being free, and Microsoft may have shot themselves in the foot yet again with their exorbitant pricing policies.
Google's launch of a new browser named Chrome this week has been met with a somewhat muted response. Although it was expected that at some point Google would launch a browser, there is still concern as to just where this new browser would fit into the market.
Firefox has for several years been taking chunks out of the dominance of the Internet Explorer, and if Google's Chrome is going to be taking users from anywhere, it will likely be those that use the likes of Firefox, Opera or Safari.
However Chrome isn't intended as a direct replacement for Firefox, or even Internet Explorer for that matter. Chrome is aimed at a completely different market. Chrome's primary aim is to give better compatibility and reliability with some of it's other services such as Google Apps.
"What we really needed was not just a browser, but also a modern platform for web pages and applications, and that's what we set out to build,"
Mr Pichai, VP Product Management.
Regardless of it's main focus, there are plenty of raised eyebrows at the thought of a Google browser.
Google does not have a good record when it comes to privacy, up until recently it kept search data indefinitely, now Google says that it would only be keeping search data for up to two years.
Many have questioned just why the search company needs to keep this data at all, let alone for two years, and the EU and Norway have launched investigations into this type of data retention.
It is quite worrying the amount of data that Google, and other search engines are able to glean from simple searches, and it is not clear whether after two years the information is in some way randomised, or deleted entirely.
Peter Fleischer, European privacy counsel for Google, has said that the company..
"...needed to keep search information for some time for security purposes – to help guard against hacking and people trying to misuse Google's advertising system."
Even so, two years is a long time to keep information on the off chance of misuse.
Provided that users leave Chrome's auto-suggest feature on and have Google as their default search provider, Google will have access to any keystrokes that are typed into the browser's Omnibox, even before a user hits enter....A Google representative told CNET News that the company plans to store about 2 percent of that data--and plans to store it along with the Internet Protocol address of the computer that typed it....In theory, that means that if one were to type the address of a site--even if they decide not to hit enter--they could leave incriminating evidence on Google's servers.
Quite a surprising feature and again we must ask if this is really necessary. There is an option (Incognito mode) that prevents the sending of information, but it is unclear how well this mode is labelled and whether the average user will be aware of it. As in all aspects of personal privacy the options should be the other way round, Incognito mode should be enabled by default and turned off by users that wish to, as the vast majority of users are likely just to use the browser as is.
There is of course another area in which Google is competing with Microsoft, the cloud. The cloud is where services are provided as web based applications, in other words where no software is purchased or downloaded, the user simply needs a web browser to use the applications. Many companies are moving into providing services in 'the cloud'; Adobe for instance provides a stripped down version of its Photoshop application for free as a web based service.
Google provides Google Apps, also for free - at least for basic use, as a web based service, directly competing with Microsoft's Office program. Admittedly the cloud appears to be a very useful way of using software at first glance. Previously those using multiple computers have to carry around flash memory cards or USB sticks containing their information and documents. Even then they had to make sure that the same software was installed on every PC they were intending to use.
Google Apps, and other services like them, make working on the move much more conveinant and remove the hassle of trying to open an important document on a PC that doesn't have Microsoft Office installed.
Along with the pros, there are a few cons; this move toward providing a service rather than the actual software means that the user has nothing tangible to rely on. Should the internet or even just the service provider fail, they are lost.
Then of course there are the costs, at present many of these services are free with premium paid for subscription services an option, but once the dominance of the likes of Microsoft is broken, what is to stop these service providers charging everyone? Moreover, what is to stop them setting whatever price they want to, once you have become tied in to their services?
Add to this the privacy issues concerning someone like Google, who have access to your search records and information; with GMail, your emails and content; with your browser, the websites you visit and your browsing habits; and with your documents and accounts they may well have filled in the last gaps in your private information.
"By submitting, posting or displaying the content you give Google a perpetual, irrevocable, worldwide, royalty-free, and non-exclusive license to reproduce, adapt, modify, translate, publish, publicly perform, publicly display, and distribute any Content which you submit, post, or display on or through, the Services."
This was of course later altered when it was pointed out, but it does bring up another important point, few people actually read EULAs and this article shows why perhaps they should do.
Google envisions a move toward the cloud in most aspects of every day computing, and in fact this view is nothing new. Bill Gates said many years ago that he believed computing would move toward a subscription service, where Microsoft are paid every month, just like other utility providers. Now such a reality is closer than ever.
However a complete move to remote computing is unlikely, what with the prevalence of cheap flash storage and with laptops and netbooks being so cheap and open source software being so freely available, there isn't a desperate need for such a solution.
Should Google resolve its privacy issues, it will be an excellent option for many people, and that of course is what is key - choice. It would give users a variety if options of how to use software, so they aren't tied to just one method, particularly those on the move. The smart people would have a laptop and/or a flash card and perhaps use Google Apps too, just in case one should fail.
Google's Chrome is an interesting move, Chrome isn't yet the answer to anyone's prayers, but it will certainly push forward browser development and open new avenues. If the fears over Google and privacy turn out to be wholly unfounded, then it may help enable a much freer computing environment for everyone.


Is Dual Citizenship Legal For Americans ?
Does FATCA Signal The Demise Of The US Dollar?
A number of current US Passport holders are of course concerned about the so-called FATCA regulations that are being imposed upon foreign banks (meaning non US banks) for reporting purposes (in the case whereby such US citizens might have accounts abroad or outside the US as a better way to say it). Considering we have received a deluge of emails from our clients over this topic, we thought it important to separate the wheat from the chaff, as they say. In addition, in our opinion, this absolute stupidity on the part of the US Treasury at a time when foreigners already are reluctant to invest in US Dollars, in US Banks or into US Government Bonds only hastens the demise of the US Dollar as a currency for trade settlement, and reserves abroad.
First and foremost it is important to understand what the regulations actually say in terms of a reporting issue for FATCA. Which is to explain that technically a reportable event occurs when the individual US citizen with the bank account abroad has more than US$50,000 at year's end in the account, and or if they had US$70,000 (or more) at any one time during the entire year in that same account. Now with that said, are the foreign banks going to write a computer program to sort out these instances or are they going to report everything regardless? Honestly, we cannot say and we cannot tell you how each individual bank around the world is going to handle it. What we can tell is that many banks, or at least the ones we have talked to, are highly perturbed and annoyed by all this. After all, it is an added expense to write new computer code to request a program for all this nonsense and the teeth of forcing compliance is really an issue that could potential effect all of that bank's clients that might have accounts in US Dollars, which are NOT US citizens.
In order to understand all this, we need to back up a minute and explain just how correspondent banking works, which is what the term is when a bank outside of the US has a sort of clearing account with a US based bank (for the purposes of being able to transact and offer USD accounts to it's own customers). Let us illustrate this idea using a fictitious bank named Palm-Tree Bank, located on the beautiful tropical island of Bogo, and Big-Bank of Chicago, located in the US.
The nice people at the Palm Tree Bank have decided to offer US Dollar savings accounts to it's own banking customers in Bogo, and to allow their customers to deposit and clear US Dollar checks, send and receive US Dollar wire transfers, and so on. However, the national currency or money used in Bogo is the Bogo Rupee. So, in order to offer accounts and transactions in another foreign currency to it's bank customers, they need to open what is known as a correspondent relationship with a bank inside the country that uses that other currency. In the case of US Dollars, that would mean Palm-Tree Bank would need to establish a correspondent account with some bank inside the United States (Big-Bank in Chicago, using our made up example). And if Palm-Tree Bank wanted to offer Euros, they would need to do the same with some bank inside of the EU. Japanese Yen, with a Japanese Bank in Japan – etc. and so on.
The type of account that Palm-Tree opens with the foreign bank is called a correspondent account, which is what can also be called an Omnibus Account or Clearing Account, just to name two terms that pretty much mean the same thing. Which is to say, an account in the name of Palm-Tree Bank containing ALL combined balances of ALL their customers. In other words, collectively all of the clients of Palm-Tree combined might have a total of US$50 Million Dollars and there is ONE correspondent account with Big-Bank in Chicago, in the name of Palm-Tree with that balance. The people at Big-Bank in Chicago have NO idea who that money belongs to, they just know they have a special correspondent account for Palm-Tree set up with them. The accounting department at Palm-Tree of course knows who is the owner of that money on an individual basis, but the US Bank does NOT have a clue. This is a very important point to understand because here is the rub.
The US Treasury Department (or the Internal Revenue Service, perhaps more precisely) wants to know what monies US citizens have abroad in a particular bank, in a particular country. So, they go to Big-Bank in Chicago and they ask: Who are all the individual US citizens banking at Palm-Tree Bank in Bogo, which we know is a correspondent customer of yours? Big-Bank in Chicago says dunno, go ask them. They do, and Palm-Tree Bank says we have very strict confidentiality laws here in Bogo and we cannot release this information to you without a court order from OUR Supreme Court and it has to be relevant to a specific client, not a witch hunt to find out who all our clients are. So, here is the AHA moment.
The US authorities get this brilliant idea that they will coerce such information from all these foreign banks abroad through the threat of confiscation (or in our opinion, the set up for a bail-in, but not of a bank but rather of the US Treasury). They pass a DOMESTIC law or regulation inside the United States telling all other countries to comply because they just passed a law inside the US that says so. Come again? So that means that Germany passes a law saying it's own citizens must wear yellow pants on Tuesday – AND they are telling Canada, Mexico, China and every other country on the planet that they MUST enforce this law inside their own respective countries IF there are any German citizens there on a Tuesday (give me what you guys are smoking because it must be really, really good stuff).
But if that is not insane and preposterous enough, the problem is the teeth or fine involved if the foreign bank does not comply OR if the US authorities think (as in let's just make some stuff up we are not really sure about) that the foreign bank is not coming clean 100 percent. And the fine is a 30 percent confiscation of the funds held in Palm-Tree's US based correspondent account with Big-Bank in Chicago (read theft, robbery, involuntary donation, or whatever other terms suits you). Now, presumably one would guess and assume (if one actually believes the law works the way it should, and fairness abounds) that they are only going to deduct the 30 percent belonging to a particular US citizen that they are sure about and are sure of his balance. But what if they are wrong (we all know the IRS never makes mistakes, excuse me while I cough up a fur ball). What if, not only are they wrong about the individual person in question being a US citizen, but wrong about the balance amount as well? What if, just for fun, they deduct 30 percent of Palm-Trees US$50 Million Dollar balance that belongs to ALL their customers, whomever and where ever they might be from? Now we are talking about a bail-in scenario – and this is why one solution would be NOT to even bother with any US Dollar accounts at all. And the reason is, despite all the rhetoric, the only thing they have access to and can confiscate are the US Dollars housed or domiciled inside the US banking system. If you have no USD, you have no assets to grab.
Now I know what you are thinking (and you would be correct) in that right now the US Dollar still is used extensively in trade settlement, etc. So, to simply say you are going to eliminate US Dollars altogether (especially if you operate a business) is very difficult to do. But we are talking about today, right now, and not maybe 6 months from now, one year from now, or three years from now. There are a number of changes taking place in the world economically and politically to indicate other currencies are going to dominate and or maybe even some kind of gold settlement as well used instead. The Chinese currency is one such monetary unit that other countries are embracing. And to highlight this point, we think it very interest and very telling that the retail arm of the Central Bank in the Dominican Republic has recently announced they will start conducting business for it's retail customers in Chinese Yuan. And if it is now starting with Banreservas (the retail banking part of the Dominican Central bank), then it certainly will eventually be rolled out to all the other private banks in the country.
With all this said, you may view these comments as being anti-American and they are NOT. It is the politicians, the corruption and the ineptitude that has created the situation today and not the individual hard working citizens and businessmen trying to get away from it all. Let us take a short walk down history lane to explain. First the US Banks throw common sense and sound business practices out the window and begin issuing mortgages with no money down and no income documentation (where were the banking regulators we might ask?). If you could fog up a mirror, you probably could have obtained a US$1 Million Dollar mortgage from a US Bank prior to 2007. Of course the banks could care less because they knew these mortgages would not stay on their books, rather to be re-sold to Wall Street firms. The Wall Street firms then packaged all these risky and unsound mortgages to individual investors, foreign banks and even foreign governments. Doing so all the while with US based credit rating agencies claiming these were AAA rated investments. But wait, it gets even better. Then, after that bubble blows up the US Federal Government and the US Federal Reserve decide it would be a wonderful idea to solve a debt problem by doing what? By having the US Federal Reserve and US Federal Government take on even more debt to prop up the foolish and insolvent US banking concerns that did all this. But how in the world can any government simply issue more debt? Easy, by creating more money out of thin air.
So, what do foreigners see in terms of the US Political and Financial system in terms of what has gone on over the past decade?
1. Many large so-called too big to fail banks have done just that, if not declaring so explicitly then implicitly. And where are the US Banking and Financial regulators? Asleep at the wheel, all inside a country declaring to the rest of the world it's superiority regarding regulation, rule of law and so on.
2. Wall Street Firms and now the banks that have bought or created their own financial units to offer stock brokerage services have been engaged in flash trading, front running and other sorts of nonsense to cheat the very customers they claim they are protecting (Read an excellent and recent book titled: Flash Boys by Michael Lewis to understand what has gone on). And we repeat: Where were the government regulators with all this going on?
3. US Securities rating agencies, promoting themselves as being the most honest and untainted sources of true information for investors had been rating sub-par quality investments as AAA, when they were not. But, the rating agency brokerage firm relationship had been corrupted by fees paid to the rating agencies by the brokerage firms, who felt compelled to rate this junk as the brokerage firm wanted (less see their income dry up and go elsewhere). And again we ask: Who is watching or rating the rating agencies?
4. And now there is a new politically acceptable paradigm on the block, affectionately known as a bail-in. And what that means is if any bank goes kaput, the local government in the country where the bank is located thinks it a wonderful idea that the bank solve it's problems by taking money away from the bank's own depositors. What's worse, right after this happened in Cyprus, a talking head from the European Central Bank and banking officials in Canada turned around to say: Yeah, what a great idea.
So, let us say you are a foreign bank watching all this take place. And remember, a foreign bank with a correspondent account in another bank located in another country for the sole purpose of being able to conduct business in that other countries currency is a customer too. This means, the bail-in thing would apply to them also. And the final cherry on top is a threat by the US Treasury to take money out of the foreign bank's account IF they do not comply with what US authorities want in terms of turning over information, even though it might be against the law to do so in the country where the foreign bank is located. What would you do? Would you want to risk it and continue using the US Dollar or offering accounts or services in US Dollars IF another viable, stable and more honest currency came along? Think about it.
© 2014 Ascot Advisory Services | Newsletter Articles Section | Feature: Is FATCA The Demise of The US Dollar?


You are far from an ordinary woman, so why should you settle for ordinary jewelry? The jewelry you wear showcases your own personality and style, yet finding what fits your mood in a way that speaks volumes about the woman you really are, and that isn't always easy. That is why I have taken my goldsmith knowledge and flare for design to step beyond the norm within the jewelry industry to strike out with a unique collection that every woman can call her own.
Talia Jewelry isn't just a collection of charms, bracelets and necklaces – it is a showcase of independent pieces that I have painstakingly designed with the express purpose of being recreated by each and every customer who orders one in order to create her own one-of-a-kind pieces. Every time you slip on a piece of Talia Jewelry, you can rest assured that no one else is going to be wearing the exact same piece.


Do there exist, in France, other statues, busts or paintings depicting Beethoven? I don't know of any more. If you have any information, don't hesitate to tell me. Thank you.
In the meantime, there exist numerous towns and cities in France which honour Beethoven in naming a street after him: Toulouse, Grenoble, Valence, Riom, Châteaubriant and Epernay are some of them.
There exist also schools named after Ludwig van Beethoven, in Montpellier and in Saint-Germain en Laye, a Beethoven gymnasium in Epernay, a retirement home in Arcachon, a centre of auditive re-education in Le Petit Quevilly (Seine Maritime), a CAMSP (Center for young people with handicaps) in Rouen, a block of flats in Nice, and a van Beethoven hotel in Saint-Julien de Peyrolas (Gard).
There is a small street in Toulouse which carries the name 'Rue Ludwig van Beethoven'.