Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll a-depleted cytoplasmic membrane in phototrophically grown cells

Parks, Lawrence C.; Niederman, Robert A.

doi:10.1016/0005-2736(78)90065-2

Cited by 43 publications

(30 citation statements)

References 32 publications

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“…The ICM is physically continuous with the cytoplasmic membrane but functionally differentiated. The components for aerobic respiration are located in the cytoplasmic membrane, while the reaction center (RC) and two light-harvesting antenna complexes responsible for photosynthetic growth are located in the ICM (30,31).…”

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Complementation of a reaction center-deficient Rhodobacter sphaeroides pufLMX deletion strain in trans with pufBALM does not restore the photosynthesis-positive phenotype

1990

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The puf operon in Rhodobacter sphaeroides is composed of the genes for the photosynthetic reaction center L and M subunits, light-harvesting antenna complex I, and one other open reading frame termed puJX.Complementation of a reaction center-deficient, photosynthetically incompetent pufLMX deletion strain in trans with a fragment containing the entire puf operon, including puJX and an additional 1,100 base pairs of DNA downstream of puJX, restored the reaction center and the photosynthesis-positive phenotype. Complementation of the same strain with puJBALM restores the reaction center to the level seen with the entire puf operon but not the photosynthesis-positive phenotype. Northern (RNA) blot analysis revealed that oxygen regulated transcription was not blocked in the absence of puJX and the downstream region. Spectroscopic and protein analyses indicated that the pigment-binding protein complexes, including the reaction center, were expressed and showed normal absorption characteristics. A 20% reduction in the amount of light-harvesting antenna complex II and a corresponding increase in the amount of light-harvesting antenna complex I were observed in the deletion strain harboring the plasmid with the puf insert lacking the puJX gene and the downstream region compared with those complemented with the entire puf operon and an additional downstream 1,100 base pairs.The purple nonsulfur bacterium Rhodobacter sphaeroides produces an extensive system of intracytoplasmic membranes (ICM) when grown photosynthetically or under conditions of low oxygen partial pressure (21). The ICM is physically continuous with the cytoplasmic membrane but functionally differentiated. The components for aerobic respiration are located in the cytoplasmic membrane, while the reaction center (RC) and two light-harvesting antenna complexes responsible for photosynthetic growth are located in the ICM (30, 31).The two light-harvesting complexes serve as antennae which capture and transfer light energy to the RC. Lightharvesting antenna complex I (LHI), characterized by a single infrared absorption maximum at 875 nm (at room temperature; 888 nm at 77K), is composed of two polypeptides, two bacteriochlorophyll (Bchl) a molecules, and two carotenoids (8). Light-harvesting antenna complex II (LHII), with two infrared absorption maxima at 800 and 850 nm, also consists of two polypeptides but contains three molecules of Bchl a and a single carotenoid (47).The RC is composed of three subunits termed H (heavy), M (medium), and L (light) on the basis of their migration properties in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The three subunits are present in a 1:1:1 stoichiometry and contain a Bchl a dimer, two monomeric molecules of Bchl a, two monomeric molecules of bacteriopheophytin a, two ubiquinones, and a nonheme iron (for a review, see reference 18). The RC undergoes reversible bleaching upon light excitation that can be measured at 860 or 600 nm. These two maxima have been assigned to the Qy and Qx transition ba...

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Complementation of a reaction center-deficient Rhodobacter sphaeroides pufLMX deletion strain in trans with pufBALM does not restore the photosynthesis-positive phenotype

1990

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“…MATERIALS AND METHODS R. sphaeroides (NCIB 8253) was grown phototrophically at 30°C essentially as described (13) with 40-W General Electric incandescent lamps at a light intensity of 1800 lux as measured on a Weston model 756 illumination meter. Chromatophores were purified from French-press extracts as described (7).…”

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Isolation and characterization of the pigment-protein complexes of Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis.

Broglie¹,

Hunter²,

Delepelaire³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

153

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When purified photosynthetic membranes from Rhodopseudomonas sphaeroides were treated with lithium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis at 4VC, up to 11 pigment-protein complexes were resolved. Absorption spectra revealed that the smallest complex contained reaction center pigments and the others contained the antenna components B850 and B875 in various proportions. Of these antenna complexes, the largest was almost entirely B850 and the smallest contained only B875. After solubilization at 1000C and electrophoresis on polyacrylamide gradient gels, the B850 complex gave rise to two polypeptide components migrating with apparent M, of 10,000 and 8000, whereas with the B875 complex, two components were observed with apparent Mr of 12,000 and 8000. The reaction center complex gave rise to only the 24 The photosynthetic apparatus of the photoheterotrophic bacterium Rhodopseudomonas sphaeroides is localized in a system of intracytoplasmic membranes (1) which, upon mechanical disruption, gives rise to vesicles termed "chromatophores" (2). These contain most of the antenna (light-harvesting) and reaction center bacteriochlorophyll a (Bchl)-protein complexes (2). Radiant energy harvested by antenna migrates to the reaction center where productive photochemistry occurs (3, 4). Absorption spectra reveal two antenna complexes designated B850 and B875 on the basis of their absorption maxima in the near-infrared (5). B875 is associated with the reaction center in a molar ratio of about 30:1 (6), unlike B850 levels which vary with changes in light intensity (6) or the stage of membrane development (7). A detailed understanding of these Bchlprotein complexes requires their isolation from the membrane. After detergent treatment, reaction center preparations have been purified from chromatophores of both the carotenoidless mutant R-26 (8, 9) and the wild type (10); similarly, the B850 complex has also been isolated from the latter (11). To our knowledge, no reports on the isolation of B875 from wild-type R. sphaeroides have appeared.Recently, a novel procedure has been developed for the isolation of chlorophyll-protein complexes from thylakoid membranes of Chlamydomonas reinhardtii and higher plants (12). After solubilization of membranes in lithium dodecyl sulfate (LiDodSO4) and polyacrylamide gel electrophoresis with this detergent at 40C, two new chlorophyll-protein complexes were isolated. Under these conditions, the dissociation of pigment from the complexes is minimized. LiDodSO4 was used because the sodium salt, previously used, precipitates in the cold. It is shown here that, in R. sphaeroides wild-type chromatophores, LiDodSO4/polyacrylamide gel electrophoresis provides a convenient procedure for the isolation of the B875-antenna complex in addition to B850 and reaction centers that lack the heavy subunit.MATERIALS AND METHODS R. sphaeroides (NCIB 8253) was grown phototrophically at 30°C essentially as described (13) with 40-W General Electric incandescent lamps at a light intensi...

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“…Some bc 1 complexes are proposed to populate the neck region of the vesicle, because it is known that a noninvaginated cytoplasmic membrane fraction can be purified from photosynthetically grown cells and that these membranes not only lack photosynthetic complexes, but are also enriched in respiratory components (58). However, other bc 1 complexes may be located equatorially closer to the RC-LH1-PufX complexes.…”

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Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle

Sener

Olsen

Hunter

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

171

333

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The photosynthetic unit (PSU) of purple photosynthetic bacteria consists of a network of bacteriochlorophyll-protein complexes that absorb solar energy for eventual conversion to ATP. Because of its remarkable simplicity, the PSU can serve as a prototype for studies of cellular organelles. In the purple bacterium Rhodobacter sphaeroides the PSU forms spherical invaginations of the inner membrane, Ϸ70 nm in diameter, composed mostly of lightharvesting complexes, LH1 and LH2, and reaction centers (RCs). Atomic force microscopy studies of the intracytoplasmic membrane have revealed the overall spatial organization of the PSU. In the present study these atomic force microscopy data were used to construct three-dimensional models of an entire membrane vesicle at the atomic level by using the known structure of the LH2 complex and a structural model of the dimeric RC-LH1 complex. Two models depict vesicles consisting of 9 or 18 dimeric RC-LH1 complexes and 144 or 101 LH2 complexes, representing a total of 3,879 or 4,464 bacteriochlorophylls, respectively. The in silico reconstructions permit a detailed description of light absorption and electronic excitation migration, including computation of a 50-ps excitation lifetime and a 95% quantum efficiency for one of the model membranes, and demonstration of excitation sharing within the closely packed RC-LH1 dimer arrays.excitation transfer ͉ network kinetics ͉ photosynthetic light harvesting ͉ quantum efficiency ͉ systems biology P hotosynthesis, the main source of energy for the biosphere (1, 2), is initiated when the thousands of pigments that cooperate to form an interconnected photosynthetic unit (PSU) harvest and transfer solar energy before its conversion to a charge separation. Peripheral pigment-protein complexes deliver energy to a reaction center (RC) (3-6), where it is used for the transmembrane electron transfers (7, 8) that eventually drive ATP synthesis. The structures of the key protein complexes involved in this process have been solved both for oxygenic photosynthetic organisms, such as cyanobacteria, algae, and plants (9-14), and for anoxygenic photosynthetic bacteria (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). This recent progress makes it possible to study the biophysical processes involved in photosynthesis in atomic detail all the way down to the quantum mechanical level (25-31). However, it remains a challenge to understand how a biological membrane comprising hundreds of photosynthetic complexes functions with great efficiency. To address this challenge we embarked on the in silico construction of an entire photosynthetic membrane, namely the purple bacterial PSU (32), based on a combination of cryo-EM (24, 33-35), NMR (22), x-ray crystallography (18,19,23,36), and atomic force microscopy (AFM) (37-41) data.The purple bacterial PSU displays remarkable simplicity compared with its eukaryotic, oxygenic analogues, being evolutionarily more primitive (42). It contains six different kinds of proteins that work cooperatively: LH2 antenna complexes (18,19,2...

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Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll a-depleted cytoplasmic membrane in phototrophically grown cells

Cited by 43 publications

References 32 publications

Complementation of a reaction center-deficient Rhodobacter sphaeroides pufLMX deletion strain in trans with pufBALM does not restore the photosynthesis-positive phenotype

Complementation of a reaction center-deficient Rhodobacter sphaeroides pufLMX deletion strain in trans with pufBALM does not restore the photosynthesis-positive phenotype

Isolation and characterization of the pigment-protein complexes of Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis.

Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle

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