In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.

Formation of oil-water emulsions during bacterial growth on hydrocarbons is often attributed to biosurfactants. Here we report the ability of certain intact bacterial cells to stabilize oil-in-water and water-in-oil emulsions without changing the interfacial tension, by inhibition of droplet coalescence as observed in emulsion stabilization by solid particles like silica.Emulsions are commonly observed when liquid hydrocarbons and water are mixed during bioremediation or fermentation (2). These emulsions dramatically increase the area of the oil-water interface, thereby enhancing bioavailability. For a dispersion of one liquid in another to be stable enough to be classified as an emulsion, a third component, such as a surfactant, must be present to stabilize the system. Fine solid particles such as silica beads can also stabilize emulsions if they attach at the interface between the oil and the water to prevent droplets from coalescing (4,14,23). While bacteria can produce surfactants or emulsifiers that stabilize emulsions (1, 5, 18), some microorganisms can emulsify hydrocarbons even in the absence of cell growth or uptake of hydrocarbons (18). The latter observation suggests that emulsification may be associated with the surface properties of the cells, as a result of attachment to the oil-water interface by general hydrophobic interactions rather than specific recognition of the substrate (5, 21). Bacterial cells may, therefore, behave as fine solid particles at interfaces.Given that fine solid particles can stabilize oil-water emulsions, our hypothesis was that intact, stationary-phase bacteria can stabilize oil-water emulsions by adhering to the oil-water interface and that this property is related to cell surface hydrophobicity. We selected four hydrocarbon-degrading bacterial species and determined the surface properties of washed stationary-phase cells by cell adhesion to hydrocarbons, the contact angle, and the interfacial tension. The structure of the emulsions was observed by confocal microscopy, and the behavior of oil droplets in bacterial suspensions was measured with a micropipette apparatus. These results were used to make general inferences about the ability of intact, bacterial cells to stabilize oil-water emulsions.The n-alkane-degrading bacteria employed in this study were Acinetobacter venetianus 20), Rhodococcus erythropolis 20S-E1-c (9), and Rhizomonas suberifaciens EB2-1a (8). Pseudomonas fluorescens LP6a degrades a range of aromatic hydrocarbons but not n-alkanes (8). All bacterial cultures were grown in Trypticase soy broth (Difco, Sparks, Md.) with incubation at 28°C and gyratory shaking. Each culture was harvested at its stationary phase by centrifugation and washed twice with 100 mM phosphate buffer (pH 7).The harvested and washed cells were used to characterize the cell surface properties. Bacterial adhesion to hydrocarbons (BATH) was measured as described by Rosenberg et al. (17). Cells were resuspended in phosphate buffer to an optical density at 600 nm (OD 600 ) of about 0.6....

Microbial adhesion to surfaces and interfaces is strongly influenced by their structure and physicochemical properties. We used atomic force microscopy (AFM) to measure the forces between chemically functionalized AFM tips and two bacterial species exhibiting different cell surface hydrophobicities, measured as the oil/water contact angle (theta): Acinetobacter venetianus RAG-1 (theta = 56.4 degrees ) and Rhodococcus erythropolis 20S-E1-c (theta = 152.9 degrees ). The forces were measured as the AFM tips, coated with either hydrophobic (octadecane) or hydrophilic (undecanol) groups, approached the bacterial cells in aqueous buffer. The experimental force curves between the two microbial cells and functionalized AFM probes were not successfully described by the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid stability. To reconcile the discrepancy between theory and experiments, two types of extended DLVO models were proposed. The first modification considers an additional acid-base component that accounts for attractive hydrophobic interactions and repulsive hydration effects. The second model considers an additional exponentially decaying steric interaction between polymeric brushes in addition to the acid-base interactions. These extended DLVO predictions agreed well with AFM experimental data for both A. venetianus RAG-1, whose surface consists of an exopolymeric capsule and pili, and R. erythropolis 20S-E1-c, whose surface is covered by an exopolymeric capsule. The extended models for the bacteria-AFM tip force-distance curves were consistent with the effects of steric interactions.

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.

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