The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.

Mutations in the tol-pal genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as tolC, lpp, and rfa mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of thetol-pal genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed intolC and rfaD cells in the same conditions. AtolA deletion in three different Escherichia coli strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction oftol strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic ''virion factory,'' we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 59 and 39 extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus ''virophage.'' These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)-will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.

Over the last 9 years, the structures of the various components of the bacterial photosynthetic apparatus or their homologues have been determined by x-ray crystallography to at least 4.8-Å resolution. Despite this wealth of structural information on the individual proteins, there remains an urgent need to examine the architecture of the photosynthetic apparatus in intact photosynthetic membranes. Information on the arrangement of the different complexes in a native system will help us to understand the processes that ensure the remarkably high quantum efficiency of the system. In this work we report images obtained with an atomic force microscope of native photosynthetic membranes from the bacterium Rhodospirillum photometricum. Several proteins can be seen and identified at molecular resolution, allowing the analysis and modeling of the lateral organization of multiple components of the photosynthetic apparatus within a native membrane. Analysis of the distribution of the complexes shows that their arrangement is far from random, with significant clustering both of antenna complexes and core complexes. The functional significance of the observed distribution is discussed. I n photosynthesis, highly efficient multiprotein assemblies convert sunlight into chemical potential energy. This process requires several different membrane proteins that funnel light energy to the primary reaction center (RC) and then ensure a cyclic electron transfer chain that converts this energy into an electrochemical potential (1) and, finally, an ATP synthase that is able to store the energy in the phosphodiester bond of ATP (2). A challenge in structural biology is to analyze the structural basis of this efficiency in native membranes. More precisely, the relationship between the different components of the system that ensure efficient energy and electron transfer needs to be determined (3, 4).In photosynthetic bacteria, a large amount of structural information about the individual components of the photosynthetic unit (PSU) is available. The PSU is an assembly made up of the RC associated with the light-harvesting proteins LH1 and LH2, containing chlorophylls and carotenoids. All components cooperate in absorbing light effectively and channeling energy to the RC. In particular, high-resolution structures of two LH2s from Rhodopseudomonas acidophila and Rhodospirillum molischianum and of two RCs from Rhodopseudomonas viridis and Rhodobacter sphaeroides are available (5-9). Electron crystallography data have revealed a hexadecameric assembly of LH1 around the RC in Rhodospirillum rubrum (10, 11). More recently, atomic force microscope (AFM) topographs of native membranes of R. viridis could be acquired. The data unambiguously reported an elliptical hexadecameric arrangement of the LH1 around each RC in a noncrystalline native environment (12). A 4.8-Å x-ray structure of the core complex of Rhodopseudomonas palustris was also elliptical, but with 15 LH1 subunits and one unidentifiable peptide subunit surrounding the RC (13). This str...

No abstract

No abstract

The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase. This pathway requires the function of a macromolecular complex called the Xcp secreton. The Xcp secreton shares many features with the machinery involved in type IV pilus assembly. More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins. We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus. Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure. Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits. Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P. aeruginosa. In addition, heterologous pseudopilins can be assembled by P. aeruginosa into a type II pseudopilus. Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities. These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections.Pseudomonas aeruginosa is a gram-negative, opportunistic bacterial pathogen that is responsible for severe nosocomial infections and is also a key agent in the early deaths of patients suffering from cystic fibrosis (17). The organism is ubiquitous; it is found in many different ecological habitats and may infect a wide variety of hosts (32). These adaptive properties can be related to the large genome size of the bacterium (6.3 Mb) (37).The pathogenicity of P. aeruginosa and its abilities to infect tissues and to colonize and establish itself on different surfaces is linked to the production of several toxins, hydrolytic enzymes, and adhesins. There are several secretory pathways that allow the extracellular release of P. aeruginosa enzymes and toxins (38). Whereas type I and type III pathways are thought to allow a one-step transport process of exoproteins across both inner and outer membranes of the cell envelope, the type II pathway drives exclusively translocation across the outer membrane (30). The translocation of the type II-dependent exoproteins across the inner membrane is achieved by either the Sec or Tat transport systems (40). For P. aeruginosa, two functional type II systems have been characterized. The Xcp system is required for secretion of exotoxin A, lipases, phospholipases C, alkaline phosphatase, or elastase (LasB) (12), and the Hxc system is required for secretion of the low-molecular-weight alkaline phosphatase LapA (1).A number of adhesins are involved in P. aeruginosa attachment. In additi...

The bacterial twin arginine translocation (Tat) pathway is capable of exporting cofactor-containing enzymes into the periplasm. To assess the capacity of the Tat pathway to export heterologous proteins and to gain information about the property of the periplasm, we fused the twin arginine signal peptide of the trimethylamine N-oxide reductase to the jellyfish green fluorescent protein (GFP). Unlike the Sec pathway, the Tat system successfully exported correctly folded GFP into the periplasm of Escherichia coli. Interestingly, GFP appeared as a halo in most cells and occasionally showed a polar localization in wild type strains. When subjected to a mild osmotic up-shock, GFP relocalized very quickly at the two poles of the cells. The conversion from the halo structure to a periplasmic gathering at particular locations was also observed with spherical cells of the ⌬rodA-pbpA mutant or of the wild type strain treated with lysozyme. Therefore, the periplasm is not a uniform compartment and the polarization of GFP is unlikely to be caused by simple invagination of the cytoplasmic membrane at the poles. Moreover, the polar gathering of GFP is reversible; the reversion was accelerated by glucose and inhibited by azide and carbonyl cyanide m-chlorophenylhydrazone, indicating an active adaptation of the bacteria to the osmolarity in the medium. These results strongly suggest a relocalization of periplasmic substances in response to environmental changes. The polar area might be the preferential zone where bacteria sense the change in the environment.The periplasmic space lies between the inner and the outer membranes of Gram-negative bacteria. A number of processes that are vital to the growth and viability of the cell occur within this compartment. Proteins residing in the periplasmic space fulfill important functions in the detection, processing, and uptake of essential nutrient substances. These proteins are exported into the periplasm mainly via two pathways: the unfolded proteins via the Sec system (1) and the folded enzymes containing redox cofactor via the Tat 1 (or Mtt) pathway (2-4).The periplasm might not be a uniformly homogenous compartment; fine structures known as Bayer patches/bridges (5) and periseptal and polar annuli (6 -9) have been described. The existence of these structures under physiological conditions is a subject of contention (10,11). Nevertheless, these structures were proposed to provide sites required for the export of outer membrane components, murein synthesis, secretion of bacteriophages, and cell divisions (12).On the other hand, polar bacterial organization was observed with a variety of bacterial species and concerns a disparate array of cellular functions (13). In addition to the well known examples of polar organelles such as flagella, pili, and stalklike appendages at the bacterial surface, accumulating evidence shows that periplasmic, inner membranous, and cytoplasmic proteins may also exhibit polar localization under certain condition. These proteins participate in various cellula...