Biochemical and structural characterisation of membrane-containing icosahedral dsDNA bacteriophages infecting thermophilic Thermus thermophilus

AbstractIcosahedral dsDNA viruses isolated from hot springs and proposed to belong to the Tectiviridae family infect the Gram-negative thermophilic Thermus thermophilus bacterium. Seven such viruses were obtained from the Promega Corporation collection. The structural protein patterns of three of these viruses, growing to a high titer, appeared very similar but not identical. The most stable virus, P23-77, was chosen for more detailed studies. Analysis of highly purified P23-77 by thin layer chromatography for neutral lipids showed lipid association with the virion. Cryo-EM based three-dimensional image reconstruction of P23-77 to 1.4 nm resolution revealed an icosahedrally-ordered protein coat, with spikes on the vertices, and an internal membrane. The capsid architecture of P23-77 is most similar to that of the archaeal virus SH1. These findings further complicate the grouping of icosahedrally-symmetric viruses containing an inner membrane. We propose a single superfamily or order with members in several viral families

Structural insight into African horsesickness virus infection

African horsesickness (AHS) is a devastating disease of horses. The disease is caused by the double-stranded RNA-containing African horsesickness virus (AHSV). Using electron cryomicroscopy and three-dimensional image reconstruction, we determined the architecture of an AHSV serotype 4 (AHSV-4) reference strain. The structure revealed triple-layered AHS virions enclosing the segmented genome and transcriptase complex. The innermost protein layer contains 120 copies of VP3, with the viral polymerase, capping enzyme, and helicase attached to the inner surface of the VP3 layer on the 5-fold axis, surrounded by double-stranded RNA. VP7 trimers form a second, T 13 layer on top of VP3. Comparative analyses of the structures of bluetongue virus and AHSV-4 confirmed that VP5 trimers form globular domains and VP2 trimers form triskelions, on the virion surface. We also identified an AHSV-7 strain with a truncated VP2 protein (AHSV-7 tVP2) which outgrows AHSV-4 in culture. Comparison of AHSV-7 tVP2 to bluetongue virus and AHSV-4 allowed mapping of two domains in AHSV-4 VP2, and one in bluetongue virus VP2, that are important in infection. We also revealed a protein plugging the 5-fold vertices in AHSV-4. These results shed light on virus-host interactions in an economically important orbivirus to help the informed design of new vaccines

Three-dimensional reconstruction of hibiscus chlorotic ringspot virus

10.1016/j.jsb.2003.10.001Journal of Structural Biology1443253-261JSBI

Hexanol-Induced Order-Disorder Transitions in Lamellar Self-Assembling Aggregates of Bacteriochlorophyll c in Chlorobium tepidum Chlorosomes

Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spacing between 2 and 3 nm. Treatment of chlorosomes with hexanol reversibly converts the aggregated chlorosome chlorophylls into a form with spectral properties very similar to that of the monomer. Although this transition has been extensively studied, the structural basis remains unclear due to variability in the obtained morphologies. Here we investigated hexanol-induced structural changes in the lamellar organization of BChl c in chlorosomes from Chlorobium tepidum by a combination of X-ray scattering, electron cryomicroscopy, and optical spectroscopy. At a low hexanol/pigment ratio, the lamellae persisted in the presence of hexanol while the short-range order and exciton interactions between chlorin rings were effectively eliminated, producing a monomer-like absorption. The result suggested that hexanol hydroxyls solvated the chlorin rings while the aliphatic tail partitioned into the hydrophobic part of the lamellar structure. This partitioning extended the chlorosome along its long axis. Further increase of the hexanol/pigment ratio produced round pigment-hexanol droplets, which lost all lamellar order. After hexanol removal the spectral properties were restored. In the samples treated under the high hexanol/pigment ratio, lamellae reassembled in small domains after hexanol removal while the shape and long-range order were irreversibly lost. Thus, all the interactions required for establishing the short-range order by self-assembly are provided by BChl c molecules alone. However, the long-range order and overall shape are imposed by an external structure, e.g., the proteinaceous chlorosome baseplate.This study was supported by the Spanish Ministry of Science and Education (Grant No. BFU2004-04914-C02-02 to J.B.A.), the Academy of Finland (Research Fellowship No. 1118462 to R.T.), the Czech Ministry of Education, Youth and Sports and Czech Science Foundation (Projects Nos. MSM0021620835 and 206/05/2739 to J.P.). Electron microscopy was carried out in the Electron Microscopy Unit of the Institute of Biotechnology, University of Helsinki.Peer reviewe

Structure of Chlorosomes from the Green Filamentous Bacterium Chloroflexus aurantiacus▿ †

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae

Lamellar Organization of Pigments in Chlorosomes, the Light Harvesting Complexes of Green Photosynthetic Bacteria

Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature. In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates. Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints. We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy. Cryoelectron microscopy images revealed dense striations ∼20 Å apart. X-ray scattering from chlorosomes exhibited a feature with the same ∼20 Å spacing. No evidence for the rod models was obtained. The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays. The diffraction data further indicate that arrays are built from BChl dimers. The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids. The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly

Internal Structure of Chlorosomes from Brown-Colored Chlorobium Species and the Role of Carotenoids in Their Assembly

Chlorosomes are the main light harvesting complexes of green photosynthetic bacteria. Recently, a lamellar model was proposed for the arrangement of pigment aggregates in Chlorobium tepidum chlorosomes, which contain bacteriochlorophyll (BChl) c as the main pigment. Here we demonstrate that the lamellar organization is also found in chlorosomes from two brown-colored species (Chl. phaeovibrioides and Chl. phaeobacteroides) containing BChl e as the main pigment. This suggests that the lamellar model is universal among green sulfur bacteria. In contrast to green-colored Chl. tepidum, chlorosomes from the brown-colored species often contain domains of lamellar aggregates that may help them to survive in extremely low light conditions. We suggest that carotenoids are localized between the lamellar planes and drive lamellar assembly by augmenting hydrophobic interactions. A model for chlorosome assembly, which accounts for the role of carotenoids and secondary BChl homologs, is presented