Quinone pathways in entire photosynthetic chromatophores of Rhodospirillum photometricum.

In photosynthetic organisms, membrane pigment-protein complexes [light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2)] harvest solar energy and convert sunlight into an electrical and redox potential gradient (reaction center) with high efficiency. Recent atomic force microscopy studies have described their organization in native membranes. However, the cytochrome (cyt) bc(1) complex remains unseen, and the important question of how reduction energy can efficiently pass from core complexes (reaction center and LH1) to distant cyt bc(1) via membrane-soluble quinones needs to be addressed. Here, we report atomic force microscopy images of entire chromatophores of Rhodospirillum photometricum. We found that core complexes influence their molecular environment within a critical radius of approximately 250 A. Due to the size mismatch with LH2, lipid membrane spaces favorable for quinone diffusion are found within this critical radius around cores. We show that core complexes form a network throughout entire chromatophores, providing potential quinone diffusion pathways that will considerably speed the redox energy transfer to distant cyt bc(1). These long-range quinone pathway networks result from cooperative short-range interactions of cores with their immediate environment.

Chromatic adaptation of photosynthetic membranes.

Many biological membranes adapt in response to environmental conditions. We investigated how the composition and architecture of photosynthetic membranes of a bacterium change in response to light, using atomic force microscopy. Despite large modifications in the membrane composition, the local environment of core complexes remained unaltered, whereas specialized paracrystalline light-harvesting antenna domains grew under low-light conditions. Thus, the protein mixture in the membrane shows eutectic behavior and can be mimicked by a simple model. Such structural adaptation ensures efficient photon capture under low-light conditions and prevents photodamage under high-light conditions.

Watching the photosynthetic apparatus in native membranes.

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-A 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.

The 8.5 A projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits.

Two-dimensional crystals from light-harvesting complex I (LHC I) of the purple non-sulfur bacterium Rhodospirillum rubrum have been reconstituted from detergent-solubilized protein complexes. Frozen-hydrated samples have been analysed by electron microscopy. The crystals diffract beyond 8 A and a projection map was calculated to 8.5 A. The projection map shows 16 subunits in a 116 A diameter ring with a 68 A hole in the centre. These dimensions are sufficient to incorporate a reaction centre in vivo. Within each subunit, density for the alpha- and the beta-polypeptide chains is clearly resolved, and the density for the bacteriochlorophylls can be assigned. The experimentally determined structure contradicts models of the LHC I presented so far.

Two-dimensional crystallization of the light-harvesting complex from Rhodospirillum rubrum.

Homogeneous detergent-solubilized B873 light-harvesting complexes from a carotenoid-less mutant of the purple non-sulfur bacterium, Rhodospirillum rubrum G9, were reassembled spontaneously into two-dimensional (2D) hexagonal arrays during extensive and controlled dialysis. As the complexes contain only 1 to 2 mol phospholipid per mol alpha beta dimer, the arrays formed by a self assembly process are primary due to protein-protein interactions. The hexagonal lattices were analyzed by negative stain electron microscopy and digital image processing. They exhibited a unit cell size of 12.3 nm, in close agreement with the particle diameter of the active photo-unit in native chromatophore membranes. The unit cell contains a central 5 nm stain-filled depression, embraced by a ring with an outer diameter of 10 nm.

Attenuated effect of oxygen on photopigment synthesis in Rhodospirillum centenum.

Rhodospirillum centenum resembles typical nonsulfur photosynthetic bacteria in a number of respects, including its ability to grow either anaerobically as a phototroph or aerobically as a heterotroph. We demonstrate, however, that R. centenum is unusual in its ability to synthesize a functional photosynthetic apparatus regardless of the presence of molecular oxygen. Aerobically expressed photopigments were shown to be functionally active, as demonstrated by the ability of heterotrophically grown cells to grow photosynthetically, without a lag, when suddenly placed under anaerobic conditions. An R. centenum mutant that has acquired the ability to repress synthesis of photopigments in the presence of oxygen was also characterized. Both the wild type and the oxygen-repressed mutant of R. centenum were found to exhibit high light intensity repression of photopigment biosynthesis. The latter result suggests that R. centenum contains separate regulatory circuits for controlling synthesis of its photochemical apparatus by light intensity and oxygen.

Genetic analysis of photosynthesis in Rhodospirillum centenum.

A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.

Rhodospirillum centenum, sp. nov., a thermotolerant cyst-forming anoxygenic photosynthetic bacterium.

A novel non-sulfur purple photosynthetic bacterium, designated Rhodospirillum centenum, was isolated from an enrichment culture designed to favor growth of anoxygenic photosynthetic N2-fixing bacteria. R. centenum grows optimally at 40-42 degrees C and has the capacity to produce cytoplasmic 'R bodies', refractile structures not observed hitherto in photosynthetic prokaryotes. The bacterium is also unusual among photosynthetic bacteria in that it forms desiccation-resistant cysts when grown aerobically in darkness with butyrate as the sole carbon source.

Protein on the cell surface of the moderately halophilic phototrophic bacterium Rhodospirillum salexigens.

A cell surface protein (Mr 68,000) of the moderately but obligately halophilic phototrophic bacterium Rhodospirillum salexigens was identified by two independent methods: first, by labeling the cell surface with radioactive iodine and lactoperoxidase, and second, by washing cells in 30% sucrose to remove proteins attached to the cell surface by ionic bonds. The identified protein very likely represents the outermost layer of the cell envelope of R. salexigens as observed by electron microscopy. The protein was isolated. Its isoelectric point was determined to be 4.4; the excess of acidic over basic amino acids was found to be 18.3 mol%; and its average hydrophobicity was 2.26 kJ per residue.

Differences in the architecture of cytoplasmic and intracytoplasmic membranes of three chemotrophically and phototrophically grown species of the Rhodospirillaceae.

Freeze-fracture faces of membranes of either chemotrophically or phototrophically grown Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodospirillum tenue were analyzed. All three species differed from each other with respect to size as well as numerical density (number per square micrometer) of intramembrane particles. In R. rubrum the number of particles on exoplasmic fracture faces of the cytoplasmic membrane stayed nearly constant (about 900 particles per microns2), but on the plasmic fracture face there were 4,700 and 6,264 particles per microns2, respectively, under chemotrophic and phototrophic conditions. The increase in number was largely a result of an enhanced occurrence of particles 10 nm in diameter. This diameter corresponds to the mean diameter of the predominant class of particles visible on the plasmic fracture faces of intracytoplasmic membrane formed under phototrophic conditions. In R. sphaeroides the number of particles on both of the fracture faces of cytoplasmic membranes stayed nearly constant. The mean diameter of articles appeared to be slightly increased under phototrophic conditions. Particles of cytoplasmic and intracytoplasmic membranes of phototrophically grown cells were of similar diameter. The number of particles, however, on plasmic fracture faces of intracytoplasmic membranes (6,674/microns2) was significantly higher than that on cytoplasmic membranes (5,708/microns2). R. tenue, on the other hand, which does not produce intracytoplasmic membranes, showed on exoplasmic fracture faces 543 and 3,765 particles per micron2 under chemotrophic and phototrophic conditions, respectively, whereas the corresponding numerical densities of plasmic fracture faces were 4,043 and 3,711 particles per microns2. The increased number of articles on exoplasmic fracture faces was mainly the result of an increased occurrence of particles with diameters greater than or equal to 10 nm. The results are interpreted to allow for the different modes of intractyoplasmic membrane development in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides, respectively.