SummaryBacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4Ј-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.

ABSTRACTAmong the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that ofPseudomonas aeruginosabiofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response ofPseudomonas putidaDOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context.

One question in the origin of life is the time at which membrane compartments came into the picture as hosts for the first forms of metabolism. If we assume the proteins and nucleic acids came first, then it is difficult to conceive how all the macromolecular components could have been entrapped at a later time in a single compartment. On the other hand, the hypothesis that metabolism originated from inside the compartment means that we would then have to conceive semipermeable, sophisticated membranes in prebiotic times, which does not appear plausible. With this study, we believe that we can offer a partial solution to this riddle, at the same time opening a new vista on the principles of the entrapment of solute in vesicles. We used cryo-TEM to study the entrapment of the protein ferritin in liposomes. The novel, surprising principle that appears is that when lipid surfaces close up in a proteincontaining solution to form vesicles, the entrapment frequency does not follow the expected Poisson distribution, but tends to assume a power-law behaviour, characterized by many "empty" vesicles (no or very little entrapped solute), and a long decreasing tail with extremely crowded vesicles. Cryo-TEM analysis shows indeed some extremely crowded liposomes adjacent to empty ones. The conclusion is that membrane closure can accumulate a remarkable number of solutes inside some compartments. The possible mechanism and relevance of this extreme local super-concentration effect for the origin of life are discussed.The spontaneous formation of lipid vesicles (liposomes) in an aqueous phase containing one or more solutes produces a heterogeneous population of liposomes in terms of solute content. Such entrapments have generally been studied by averaging techniques, such as batch absorbance or fluorescence, whereas little attention has been devoted to studying individual encapsulation.[1] This is partly due to the technical difficulty of directly counting molecules inside liposomes. The encapsulation of biomacromolecules inside liposomes, on the other hand, is an important issue in origins of life research (protocell models) as well as in recent studies on synthetic cells. [2] A series of recent experiments within our project on the construction of minimal living cells [3] revealed possible deviations from the number of macromolecules expected to be entrapped inside liposomes of diameter d < 200 nm. In particular, with the aim of producing green fluorescent protein (GFP) inside liposomes, we prepared liposomes in the presence of the transcription-translation macromolecular machinery, namely E. coli extracts as well as PURESYSTEM [4] (a cell-free protein synthesis kit containing 36 purified components, t-RNAs, ribosomes, for a total of about 80 different macromolecules). We showed that GFP was synthesised inside liposomes, despite of the fact that the Poisson probability of liposome co-entrapment of about 80 different macromolecules (each at a concentration of 0.1-1 mm) is vanishingly small (~10 À26). In order to explain the obs...

The temperature-dependent self-assembly of the single-chain bolaamphiphile dotriacontan-1,1'-diyl-bis[2-(trimethylammonio)ethyl phosphate] (PC-C32-PC) was investigated by transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), X-ray scattering, rheological measurements, and dynamic light scattering (DLS). At room temperature this compound, in which two phosphocholine headgroups are connected by a C(32) alkyl chain, proved to be capable of gelling water very efficiently by forming a dense network of nanofibers (Kohler et al. Angew. Chem., Int. Ed. 2004, 43, 245). A specific feature of this self-assembly process is that it is not driven by hydrogen bonds but solely by hydrophobic interactions of the long alkyl chains. The nanofibers have a thickness of roughly the molecular length and show a helical superstructure. A model for the molecular structure of the fibrils which considers the extreme constitution of the bolaamphiphile is proposed. Upon heating the suspensions three different phase transitions can be detected. Above 49 degrees C, the temperature of the main transition where the alkyl chains become "fluid", a clear low-viscosity solution is obtained due to a breakdown of the fibrils into smaller aggregates. Through mechanical stress the gel structure can be destroyed as well, indicating a low stability of these fibers. The gel formation is reversible, but as a drastic rearrangement of the molecules takes place, metastable states occur.

Colloidal suspensions of triglycerides are under investigation as potential drug carrier systems. The properties of the matrix lipids are altered in the nanoparticles compared to those of the bulk material. For instance, the metastable alpha-modification of the triglycerides usually transforms into the stable beta-polymorph quite rapidly in the colloidal particles. Recently, it was observed that the alpha-modification can be preserved for a considerable period of time in tristearin nanoparticles when the particles are stabilized with a blend of saturated long-chain phospholipids and the bile salt sodium glycocholate [Bunjes, H.; Koch, M. H. J. J. Controlled Release 2005, 107, 229-243]. As triglyceride nanoparticles in the alpha-modification may offer some advantages over those in the beta-form with regard to drug delivery applications, the structure of the corresponding dispersions was investigated in more detail with differential scanning calorimetry, X-ray diffraction, and electron microscopy. The electron microscopic investigations confirmed a platelet-like, layered structure for particles in the beta-modification and revealed a spheroidal shape with concentric layers for larger particles in the alpha-form. For the first time, not only was information on the internal structure of solid triglyceride nanoparticles obtained from freeze-fracture electron micrographs but also details were observed by cryoelectron microscopy.