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...

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The ATP synthase is a nanometric rotary machine that uses a transmembrane electrochemical gradient to form ATP. The structures of most components of the ATP synthase are known, and their organization has been elucidated. However, the supramolecular assembly of ATP synthases in biological membranes remains unknown. Here we show with submolecular resolution the organization of ATP synthases in the yeast mitochondrial inner membranes. The atomic force microscopy images we have obtained show how these molecules form dimers with characteristic 15 nm distance between the axes of their rotors through stereospecific interactions of the membrane embedded portions of their stators. A different interaction surface is responsible for the formation of rows of dimers. Such an organization elucidates the role of the ATP synthase in mitochondrial morphology. Some dimers have a different morphology with 10 nm stalk-to-stalk distance, in line with ATP synthases that are accessible to IF 1 inhibition. Rotation torque compensation within ATP synthase dimers stabilizes the ATP synthase structure, in particular the stator-rotor interaction. Data analysis All image treatment and analysis of AFM topographs were performed using custom-written routines for the ImageJ image processing package (23,24) and IGOR PRO (Wavemetrics, Portland, OR).

SummaryOf considerable interest in the biology of pathogenic bacteria are the mechanisms of intercellular signalling that can lead to the formation of persistent infections. In this article, we have examined the intracellular behaviour of a Pseudomonas aeruginosa quorum sensing regulator RhlR believed to be important in this process. We have further examined the modulation of this behaviour in response to various auto-inducers. For these measurements, we have developed an assay based on the fluorescence anisotropy of EGFP fusion proteins that we use to measure protein-protein interactions in vivo . We show that the transcriptional regulator, RhlR, expressed as an EGFP fusion protein in Escherichia coli , forms a homodimer. This homodimer can be dissociated into monomers by the auto-inducer N -(3-oxododecanoyl)-L -homoserine lactone (3O-C12-HSL) whereas N -(butanoyl)-L -homoserine lactone (C4-HSL) has little effect. These observations are of particular interest as RhlR modulation of gene expression depends on the presence of C4-HSL, whereas 3O-C12-HSL modulates the expression of genes regulated by LasR. These observations thus provide a framework for understanding the regulatory network that links the various different QS regulators in P. aeruginosa . Furthermore, the technique we have developed should permit the study of numerous protein/protein or protein/nucleic acid interactions in vivo and so shed light on natural protein function.

The chromatophores of Rhodobacter (Rb.) sphaeroides represent a minimal bio-energetic system, which efficiently converts light energy into usable chemical energy. Despite extensive studies, several issues pertaining to the morphology and molecular architecture of this elemental energy conversion system remain controversial or unknown. To tackle these issues, we combined electron microscope tomography, immuno-electron microscopy and atomic force microscopy. We found that the intracellular Rb. sphaeroides chromatophores form a continuous reticulum rather than existing as discrete vesicles. We also found that the cytochrome bc1 complex localizes to fragile chromatophore regions, which most likely constitute the tubular structures that interconnect the vesicles in the reticulum. In contrast, the peripheral light-harvesting complex 2 (LH2) is preferentially hexagonally packed within the convex vesicular regions of the membrane network. Based on these observations, we propose that the bc1 complexes are in the inter-vesicular regions and surrounded by reaction center (RC) core complexes, which in turn are bounded by arrays of peripheral antenna complexes. This arrangement affords rapid cycling of electrons between the core and bc1 complexes while maintaining efficient excitation energy transfer from LH2 domains to the RCs.

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Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.