Effects of the protonophore carbonyl-cyanide m-chlorophenylhydrazone on intracytoplasmic membrane assembly in Rhodobacter sphaeroides

AbstractThe effect of carbonyl-cyanide m-chlorophenyl-hydrazone (CCCP) on intracytoplasmic membrane (ICM) assembly was examined in the purple bacterium Rhodobacter sphaeroides. CCCP blocks generation of the electrochemical proton gradient required for integral membrane protein insertion. ICM formation was induced for 8h, followed by a 4-h exposure to CCCP. Measurements of fluorescence induction/relaxation kinetics showed that CCCP caused a diminished quantum yield, a cessation in expansion of the functional absorption cross-section and a 4- to 10-fold slowing in the electron transfer turnover rate. ICM vesicles (chromatophores) and an upper-pigmented band (UPB) containing ICM growth initiation sites, were isolated and subjected to clear-native electrophoresis. Proteomic analysis of the chromatophore gel bands indicated that CCCP produced a 2.7-fold reduction in spectral counts in the preferentially assembled light-harvesting 2 (LH2) antenna, while the RC-LH1 complex, F1FO-ATPase and pyridine nucleotide transhydrogenase decreased by 1.7–1.9-fold. For 35 soluble enzymes, the ratio of 0.99 for treated/control proteins demonstrated that protein synthesis was unaffected by CCCP, suggesting that the membrane complex decline arose from the turnover of unassembled apoproteins. In the UPB fraction, an ~2-fold accumulation was observed for the preprotein translocase SecY, the SecA translocation ATPase, SecD and SecF insertion components, and chaperonins DnaJ and DnaK, consistent with the possibility that these factors, which act early in the assembly process, have accumulated in association with nascent polypeptides as stabilized assembly intermediates

The native architecture of a photosynthetic membrane

In photosynthesis, the harvesting of solar energy and its subsequent conversion into a stable charge separation are dependent upon an interconnected macromolecular network of membrane-associated chlorophyll–protein complexes. Although the detailed structure of each complex has been determined, the size and organization of this network are unknown. Here we show the use of atomic force microscopy to directly reveal a native bacterial photosynthetic membrane. This first view of any multi-component membrane shows the relative positions and associations of the photosynthetic complexes and reveals crucial new features of the organization of the network: we found that the membrane is divided into specialized domains each with a different network organization and in which one type of complex predominates. Two types of organization were found for the peripheral light-harvesting LH2 complex. In the first, groups of 10–20 molecules of LH2 form light-capture domains that interconnect linear arrays of dimers of core reaction centre (RC)–light-harvesting 1 (RC–LH1–PufX) complexes; in the second they were found outside these arrays in larger clusters. The LH1 complex is ideally positioned to function as an energy collection hub, temporarily storing it before transfer to the RC where photochemistry occurs: the elegant economy of the photosynthetic membrane is demonstrated by the close packing of these linear arrays, which are often only separated by narrow 'energy conduits' of LH2 just two or three complexes wide

Updated guidance on the management of COVID-19:from an American Thoracic Society/European Respiratory Society coordinated International Task Force (29 July 2020)

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome-coronavirus-2. Consensus suggestions can standardise care, thereby improving outcomes and facilitating future research. METHODS: An International Task Force was composed and agreement regarding courses of action was measured using the Convergence of Opinion on Recommendations and Evidence (CORE) process. 70% agreement was necessary to make a consensus suggestion. RESULTS: The Task Force made consensus suggestions to treat patients with acute COVID-19 pneumonia with remdesivir and dexamethasone but suggested against hydroxychloroquine except in the context of a clinical trial; these are revisions of prior suggestions resulting from the interim publication of several randomised trials. It also suggested that COVID-19 patients with a venous thromboembolic event be treated with therapeutic anticoagulant therapy for 3 months. The Task Force was unable to reach sufficient agreement to yield consensus suggestions for the post-hospital care of COVID-19 survivors. The Task Force fell one vote shy of suggesting routine screening for depression, anxiety and post-traumatic stress disorder. CONCLUSIONS: The Task Force addressed questions related to pharmacotherapy in patients with COVID-19 and the post-hospital care of survivors, yielding several consensus suggestions. Management options for which there is insufficient agreement to formulate a suggestion represent research priorities.status: Published onlin

What We Are Learning from the Diverse Structures of the Homodimeric Type I Reaction Center-Photosystems of Anoxygenic Phototropic Bacteria

A Type I reaction center (RC) (Fe-S type, ferredoxin reducing) is found in several phyla containing anoxygenic phototrophic bacteria. These include the heliobacteria (HB), the green sulfur bacteria (GSB), and the chloracidobacteria (CB), for which high-resolution homodimeric RC-photosystem (PS) structures have recently appeared. The 2.2-Å X-ray structure of the RC-PS of Heliomicrobium modesticaldum revealed that the core PshA apoprotein (PshA-1 and PshA-2 homodimeric pair) exhibits a structurally conserved PSI arrangement comprising five C-terminal transmembrane α-helices (TMHs) forming the RC domain and six N-terminal TMHs coordinating the light-harvesting (LH) pigments. The Hmi. modesticaldum structure lacked quinone molecules, indicating that electrons were transferred directly from the A0 (81-OH-chlorophyll (Chl) a) acceptor to the FX [4Fe-4S] component, serving as the terminal RC acceptor. A pair of additional TMHs designated as Psh X were also found that function as a low-energy antenna. The 2.5-Å resolution cryo-electron microscopy (cryo-EM) structure for the RC-PS of the green sulfur bacterium Chlorobaculum tepidum included a pair of Fenna–Matthews–Olson protein (FMO) antennae, which transfer excitations from the chlorosomes to the RC-PS (PscA-1 and PscA-2) core. A pair of cytochromes cZ (PscC) molecules was also revealed, acting as electron donors to the RC bacteriochlorophyll (BChl) a’ special pair, as well as PscB, housing the [4Fe-4S] cluster FA and FB, and the associated PscD protein. While the FMO components were missing from the 2.6-Å cryo-EM structure of the Zn- (BChl) a’ special pair containing RC-PS of Chloracidobacterium thermophilum, a unique architecture was revealed that besides the (PscA)2 core, consisted of seven additional subunits including PscZ in place of PscD, the PscX and PscY cytochrome c serial electron donors and four low mol. wt. subunits of unknown function. Overall, these diverse structures have revealed that (i) the HB RC-PS is the simplest light–energy transducing complex yet isolated and represents the closest known homolog to a common homodimeric RC-PS ancestor; (ii) the symmetrically localized Ca2+-binding sites found in each of the Type I homodimeric RC-PS structures likely gave rise to the analogously positioned Mn4CaO5 cluster of the PSII RC and the TyrZ RC donor site; (iii) a close relationship between the GSB RC-PS and the PSII Chl proteins (CP)43 and CP47 was demonstrated by their strongly conserved LH-(B)Chl localizations; (iv) LH-BChls of the GSB-RC-PS are also localized in the conserved RC-associated positions of the PSII ChlZ-D1 and ChlZ-D2 sites; (v) glycosylated carotenoids of the GSB RC-PS are located in the homologous carotenoid-containing positions of PSII, reflecting an O2-tolerance mechanism capable of sustaining early stages in the evolution of oxygenic photosynthesis. In addition to the close relationships found between the homodimeric RC-PS and PSII, duplication of the gene encoding the ancestral Type I RC apoprotein, followed by genetic divergence, may well account for the appearance of the heterodimeric Type I and Type II RCs of the extant oxygenic phototrophs. Accordingly, the long-held view that PSII arose from the anoxygenic Type II RC is now found to be contrary to the new evidence provided by Type I RC-PS homodimer structures, indicating that the evolutionary origins of anoxygenic Type II RCs, along with their distinct antenna rings are likely to have been preceded by the events that gave rise to their oxygenic counterparts

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase.

International audienceAtomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization

The long-range organization of a native photosynthetic membrane

Photosynthesis relies on the delicate interplay between a specific set of membrane-bound pigment–protein complexes that harvest and transport solar energy, execute charge separation, and conserve the energy. We have investigated the organization of the light-harvesting (LH) and reaction-center (RC) complexes in native bacterial photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides by using polarized light spectroscopy, linear dichroism (LD) on oriented membranes. These LD measurements show that in native membranes, which contain LH2 as the major energy absorber, the RC–LH1–PufX complexes are highly organized in a way similar to that which we found previously for a mutant lacking LH2. The relative contribution of LH1 and LH2 light-harvesting complexes to the LD spectrum shows that LH2 preferentially resides in highly curved parts of the membrane. Combining the spectroscopic data with our recent atomic force microscopy (AFM) results, we propose an organization for this photosynthetic membrane that features domains containing linear arrays of RC–LH1–PufX complexes interspersed with LH2 complexes and some LH2 found in separate domains. The study described here allows the simultaneous assessment of both global and local structural information on the organization of intact, untreated membranes