Membranes of Rhodopseudomonas sphaeroides. IV. Assembly of chromatophores in low-aeration cell suspensions.

Chromatophore membrane formation was induced in low-aeration suspensions of Rhodopseudomonas sphaeroides and highly purified chromatophore preparations were isolated at various intervals between 4 and 18 h. The levels of several functional components associated with the isolated strucures were investigated. B-875, the light-harvesting bacteriochlorophyll complex associated with the reaction center, was preferentially inserted into the chromatophore membrane during the early stages of induction, and thereafter its levels reached a steady state; b- and c-type cytochromes were also maintained at essentially constant levels. In contrast, the levels of B-850, the accessory light-harvesting bacteriochlorophyll, together with its associated protein, continued to increase throughout the induction process. Increases in the levels of the major carotenoid component followed a similar course. These findings are consistent with a stepwise assembly mechanism for associated bacteriochlorophyll and protein components and suggest that separate regulatory mechanisms control the levels of functionally essential and accessory components within the membrane.     

Cell-cycle-specific fluctuation in cytoplasmic membrane composition in aerobically grown Rhodospirillum rubrum.

Aerobic growth with synchronous cell division was induced in Rhodospirillum rubrum by starvation methods. Cells were harvested at different points in the cell cycle. Analysis of the composition of the cell envelope prepared by differential centrifugation or density gradient-purified cytoplasmic membrane obtained from cells at different times indicated that the protein/phospholipid ratio fluctuated with the cell cycle. The protein/phospholipid ratio of cell envelope from selection-synchronized cells also fluctuated with the cell cycle. These studies indicate that the phenomenon of cell-cycle-dependent fluctuation in membrane composition is not restricted to the intracytoplasmic chromatophore membrane of phototrophic cells.     

Changes in the acyl lipid composition of photosynthetic bacteria grown under photosynthetic and non-photosynthetic conditions

The acyl lipids and their constituent fatty acids were studied in the photosynthetic bacteria Rhodospirillum rubrum, Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides, which were grown under photosynthetic and non-photosynthetic conditions. The major lipids were found to be phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in each bacterium. The two Rhodopseudomonas species also contained significant quantities of phosphatidylcholine. Other acyl lipids accounted for less than 10% of the total. On changing growth conditions from non-photosynthetic to photosynthetic a large increase in the relative proportion of phosphatidylglycerol was seen at the expense of phosphatidyl-ethanolamine. In Rhodospirillum rubrum the fatty acids of the major phospholipids showed an increase in the proportion of palmitate and stearate and a decrease in palmitoleate and vaccenate on changing growth conditions to photosynthetic. In contrast, the exceptionally high levels (>80%) of vaccenate in individual phospholipids of Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides were unaffected by changing growth conditions to photosynthetic. Analysis of the lipids of chromatophores, isolated from the three bacteria, showed that these preparations were enriched in phosphatidylglycerol. The large increase in this phospholipid, seen during growth under photosynthetic conditions, appeared, therefore, to be due to a proliferation of chromatophore membranes. Possible roles for acyl lipids in the formation and function of the photosynthetic apparatus of bacteria are discussed.     

Membranes of Rhodopseudomonas spheroides: Interactions of Chromatophores with the Cell Envelope

Under carefully controlled ionic conditions, large-scale preparations of highly purified chromatophores and cell envelopes were obtained from phototrophically grown Rhodopseudomonas spheroides by zonal ultracentrifugation. The majority of the bacteriochlorophyll a was located in a single, discrete chromatophore band, whereas the envelopes were nearly devoid of photopigment. The envelope fraction contained substantial quantities of succinic dehydrogenase and cytochromes, confirming that phototrophically grown cells contain a photopigment-deficient cytoplasmic membrane. Magnesium at concentrations of 1.0 mM or higher caused chromatophores to reversibly aggregate with the cell envelope. Significant aggregation was also promoted by other divalent metals (Co(2+) > Mn(2+) > Ca(2+) > Mg(2+)), but aggregation was less extensive with monovalent cations. These results account for the distribution of photopigments in two bands reported by others and further suggest that the photosynthetic apparatus of R. spheroides is located on membranes largely distinct from the cell wall-cytoplasmic membrane complex.     

Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the use of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane subproteome in response to nitrogen availability.     

Construction, characterization, and complementation of Rhodospirillum rubrum puf region mutants.

Rhodospirillum rubrum is a facultatively phototrophic bacterium that, under certain growth conditions, forms an intracytoplasmic chromatophore membrane (ICM) housing the photochemical apparatus. The puf operon of R. rubrum encodes protein subunits of the photochemical reaction center and the B880 light-harvesting antenna complex. Mutant strains of R. rubrum were constructed by interposon mutagenesis through which a kanamycin resistance gene cartridge was inserted into restriction sites and in place of restriction fragments of the puf region. Southern blot analysis demonstrated that the defective copies of puf sequences had replaced their normal chromosomal counterparts through homologous recombination. The phenotypes of the mutant strains were evaluated on the basis of puf gene expression, spectral analysis, pigment content of membranes, and electron-microscopic examination of thin sections of cells grown under semi-aerobic and dark anaerobic conditions. Alterations of the puf region affect phototrophic competence and the formation of the ICM. The latter result implies an obligatory role for puf gene products in ICM formation in R. rubrum. One mutant with a deletion in puf structural genes was complemented in trans to the wild-type phenotype. Other mutants could be restored to the wild-type phenotype only by recombination.     

Membranes of Rhodopseudomonas sphaeroides. IV. Assembly of chromatophores in low-aeration cell suspensions

Chromatophore membrane formation was induced in low-aeration suspensions of Rhodopseudomonas sphaeroides and highly purified chromatophore preparations were isolated at various intervals between 4 and 18 h. The levels of several functional components associated with the isolated structures were investigated. B-875, the light-harvesting bacteriochlorophyll complex associated with the reaction center, was preferentially inserted into the chromatophore membrane during the early stages of induction, and thereafter its levels reached a steady state; b- and c-type cytochromes were also maintained at essentially constant levels. In contrast, the levels of B-850, the accessory light-harvesting bacteriochlorophyll, together with its associated protein, continued to increase throughout the induction process. Increases in the levels of the major carotenoid component followed a similar course. These findings are consistent with a stepwise assembly mechanism for associated bacteriochlorophyll and protein components and suggest that separate regulatory mechanisms control the levels of functionally essential and accessory components within the membrane.     

Assembly of plasmid DNA and chromatophore inRhodospirillum rubrum

Summary Rhodospirillum rubrum, a photosynthetic bacterium, contains many photosynthetic vesicular membranous structures called chromatophores. The organism contains a 55 kb specific plasmid which is essential for photosynthesis, but the exact relationship between the chromatophore and the plasmid is uncertain. In this study we examined the precise localization of the plasmids, especially in relation to the chromatophores. Fluorescence in situ hybridization indicated that there are several copies of the plasmid per cell and that some plasmids are localized close to the cellular envelope. In situ hybridization at the electron-microscopic level further revealed that the plasmid localized to the periphery of the chromatophore close to the envelope. Moreover, when the chromatophore fraction was purified from cells, the plasmid DNA was observed as a cluster around the chromatophore vesicles. The assembly of the plasmid and chromatophore may be related to chromatophore formation by invagination of cell membrane.     

Membranes of Rhodospirillum rubrum: physicochemical properties of chromatophore fractions isolated from osmotically and mechanically disrupted cells.

Isolation of highly purified membrane fractions from phototrophically grown Rhodospirillum rubrum was achieved by velocity and isopyknic sedimentation under carefully controlled ionic conditions. Bacteriochlorophyll-rich and succinic dehydrogenase-rich chromatophores that were essentially devoid of contamination by non-chromatophore protein were separated from a denser fraction in extracts disrupted in a French pressure cell. Highly purified chromatophores and a nearly photopigment-free envelope fraction were also obtained from cells lysed by treatment with ethylenediaminetetraacetate-lysozyme-Brij 58. After lysis with lysozyme and ethylenediaminetetraacetate alone, about 50% of the total photosynthetic pigment was released in chromatophores similar to those isolated by the above procedures. Chromatophores prepared by each method were found to have very similar near-infrared absorption spectra, overall chemical composition, equilibrium buoyant densities in CsCl, and protein patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles of the dense, outer membrane-rich fractions were different from those of the chromatophores. The release of much of the photosynthetic apparatus as discrete chromatophores is osmotically lysed extracts necessitates a reevaluation of the concept that isolated chromatophores arise only from mechanical comminution of a larger membrane structure.     

Cell-cycle-specific oscillation in the composition of chromatophore membrane in Rhodospirillum rubrum.

Synchrony in phototrophic cultures of Rhodospirillum rubrum was induced by stationary-phase cycling or by alterations in light intensity. Intracytoplasmic chromatophore membranes were prepared by differential centrifugation. Analysis of the composition of chromatophores obtained from cells at different times indicated that the protein/bacteriochlorophyll a ratio was constant throughout the cell cycle but that the protein/phospholipid ratio oscillated. This cell-cycle-dependent fluctuation in chromatophore membrane composition was reflected in the buoyant densities of the isolated chromatophores.     

The relation between the soluble factor associated with H(+)-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli.

Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H(+)-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Ths) and a membrane-bound component are together required for activity. Ths was selectively removed from chromatophore membranes of Rhs. rubrum and was purified to homogeneity by precipitation with (NH4)2SO4 and ion-exchange, affinity dye and gel exclusion chromatography. The latter indicated an Mr of approx. 74,000 under non-denaturing conditions but analysis of the pure protein by SDS-PAGE revealed a single polypeptide, Mr 43,000. Antibodies against this polypeptide inhibited transhydrogenase activity of chromatophores and decreased the capacity of Ths to restore activity to depleted membranes. They reacted with a polypeptide of Mr 43,000 in crude cell extract, chromatophore membranes and chromatophore washings but not with transhydrogenase polypeptides from the membranes of E. coli, Rb. capsulatus or animal mitochondria. The N-terminal amino acid sequence of the 43,000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the alpha subunit of the E. coli protein. The break between the alpha and beta polypeptides of E. coli transhydrogenase is such that both components are membrane-associated. In contrast, these results suggest that in the Rhs. rubrum enzyme Ths has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble. There is a predicted similarity between Ths and the reported sequence of alanine dehydrogenase from Bacillus but Ths did not have any alanine dehydrogenase activity.     

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. I. Diffraction pattern of the photoreaction unit isolated from Rhodospirillum rubrum chromatophore and some characteristics of the structure

The X-ray diffraction pattern from chromatophore membranes of a photosynthetic bacterium, Rhodospirillum rubrum, indicates that a highly organized protein assembly exists in the membrane. The X-ray scatterer was solubilized from chromatophores by a mixture of cholate and deoxycholate. The basic component was identified as the photoreaction unit, which consists of light-harvesting bacteriochlorophyll proteins and a reaction center. The radial autocorrelation function, calculated directly from the X-ray intensity dats, made it possible to deduce certain structural features of the X-ray scatterer. 1. The maximum dimension of the X-ray scatterer is estimated to be 110-130 A. 2. The arrangement of the units in the chromatophore membrane is random. 3. Protein molecules in the unit form a rigid structure, being arranged mutually in fixed positions to give a distinct X-ray diffraction pattern. 4. The most probable structure is one which has rotational symmetry.     

Transformation of Kluyveromyces lactis by killer plasmid DNA

A single plasmid of 55 kilobases was found in crude cell lysates of each of nine strains of Rhodospirillum rubrum. Restriction endonuclease analysis showed identical fragment patterns with a given nuclease for all plasmids except one, for which an additional EcoRI site was observed. Elimination of the plasmid required that the cells be passaged several times in 25 mM calcium-containing medium, followed by at least two passages under photosynthetic growth conditions in low-calcium medium before treatment with ethyl methanesulfonate. The resulting plasmidless mutants only grew aerobically and were all incapable of pigment formation and photosynthetic growth, suggesting that plasmid DNA is required for photosynthetic competence in R. rubrum.     

Efficiency of light-driven metabolite transport in the photosynthetic bacterium Rhodospirillum rubrum.

An evaluation of the efficiency of the L-alanine and L-malate transport systems was undertaken with the photosynthetic bacterium Rhodospirillum rubrum grown on the amino acid whose uptake was measured. An all-glass apparatus was constructed for measuring transport activity under anaerobic conditions. L-Alanine transport activity decreased under conditions of Mg2+ depletion. When cells were allowed to become inactive by suspending them in the dark in Mg2+-free buffer, full activity could be restored with a few minutes by adding 20 mM Mg2+ and illuminating the cells. The transport activity was completely inhibited by carbonyl cyanide m-trifluoromethoxyphenylhydrazone and by ammonia. The quantum yield for the uptake of either L-alanine or L-malate was 0.015 molecules per photon. The results are discussed in relation to the expected efficiencies for metabolite transport and regulation by Mg2+.     

Study of electrogenic electron transfer steps in chromatophore membrane of Chromatium vinosum by the response of merocyanin dye

1. Electrogenic steps in photosynthetic cyclic electron transport in chromatophore membrane of Chromatium vinosum were studied by measuring absorption changes of added merocyanin dye and of intrinsic carotenoid.

2. The change in dye absorbance was linear with the membrane potential change induced either by light excitation or by application of diffusion potential by adding valinomycin in the presence of K⁺ concentration gradient.

3. It was estimated that chromatophore membrane became 40–60 mV and 110–170 mV inside positive upon single and multiple excitations with single-turnover flashes, respectively, from the responses of the dye and the carotenoid.

4. Electron transfers between cytochrome c-555 or c-552 and reaction center bacteriochlorophyll dimer (BChl₂) and between BChl₂ and the primary electron acceptor were concluded to be electrogenic from the redox titration of the dye response.

5. No dye response which corresponded to the change of redox level of cytochrome b was observed in the titration curve. Addition of antimycin A slightly decreased the dye response.

6. The dye response was decreased under phosphorylating conditions.

7. From the results obtained localization of the electron transfer components in chromatophore membrane is discussed.     

Phototaxis and membrane potential in the photosynthetic bacterium Rhodospirillum rubrum.

Cells of the photosynthetic bacterium Rhodospirillum rubrum cultivated anaerobically in light show phototaxis. The behavior of individual cells in response to the phenomenon is reversal(s) of the swimming direction when the intensity of the light available to them abruptly decreases. The tactic response was inhibited by antimycin, an inhibitor of the photosynthetic electron transfer system. The inhibitory effect of antimycin was overcome by phenazine methosulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic response; however, valinomycin or potassium alone had no effect. A change in membrane potential of the cells was measured as an absorbance change of carotenoid. Changes in the membrane potential caused by "on-off" light were prevented by antimycin and by valinomycin plus potassium, but not by antimycin plus phenazine methosulfate nor valinomycin or potassium alone. The results indicated that the phototactic response of R. rubrum is mediated by a sudden change in electron flow in the photosynthetic electron transfer system, and that the membrane potential plays an important role in manifestation of the response.     

Role of apparent membrane growth initiation sites during photosynthetic membrane development in synchronously dividing Rhodopseudomonas sphaeroides.

Sites of intracytoplasmic membrane growth and temporal relations in the assembly of photosynthetic units were examined in synchronously dividing Rhodopseudomonas sphaeroides cells. After rate-zone sedimentation of cell-free extracts, apparent sites of initiation of intracytoplasmic membrane growth formed an upper pigmented band that sedimented more slowly than the intracytoplasmic membrane-derived chromatophore fraction. Throughout the cell cycle, the levels of the peripheral B800-850 light-harvesting pigment-protein complex relative to those of the core B875 complex in the upper pigmented fraction were only about half those of chromatophores. Pulse-labeling studies with L-[35S]methionine indicated that the rates of assembly of proteins in the upper pigmented fraction were much higher than those of chromatophores throughout the cell cycle; rates for the reaction center polypeptides were estimated to be approximately 3.5-fold higher than in chromatophores when the two membrane fractions were equalized on a protein basis. In pulse-chase studies, radioactivity of the reaction center and B875 polypeptides increased significantly in chromatophores and decreased in the upper pigmented band during cell division. These data suggest that the B875 reaction center cores of the photosynthetic units are inserted preferentially into sites of membrane growth initiation isolated in the upper pigmented band and that the incomplete photosynthetic units are transferred from their sites of assembly into the intracytoplasmic membrane during cell division. These results suggested further that B800-850 is added directly to the intracytoplasmic membrane throughout the cell cycle.     

Kinetics and yields of bacteriochlorophyll fluorescence: redox and conformation changes in reaction center of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Induction of the bacteriochlorophyll fluorescence under rectangular shape of intense laser diode illumination (1 W cm(-2), 808 nm) was measured over wide time range from 10 mus to 4 s in whole cells, chromatophore and isolated reaction center protein of wild type and carotenoid-less mutant (R-26.1) of purple photosynthetic bacterium Rhodobacter sphaeroides. While the antenna-containing species showed large and positive variable fluorescence (F (v)) to initial fluorescence (F (0)) (F (v)/F (0) approximately 4.5 in whole cells), the isolated RC had negative change (F (v)/F (0) approximately -0.6) during photochemistry. In chromatophore from R-26.1, only seven times higher rate was measured than in isolated reaction center under identical experimental conditions. The enhancement effect of large antenna on the rate of photochemistry in chromatophore was partially compensated by the favorable pigment absorption properties in isolated RC. The transition from membrane bound to isolated form of the reaction center was probed by titration of zwitterionic detergent LDAO in chromatophore, and at 0.03% LDAO concentration, sharp change of the variable fluorescence was observed. The sudden drop was explained by the formation of LDAO micelles. After the photochemical phase, additional change of fluorescence yield could be observed in isolated RC considered as manifestation of long-living conformations of the trapped redox states of the protein characterized by non-exponential kinetics. Strong support was provided for use of the fluorescence induction to track structural and conformation changes at their earliest phases in chromatophores and isolated reaction centers.     

Development and growth of photosynthetic membranes of Rhodospirillum rubrum

In cell-free extracts from low-aeration suspensions of Rhodospirillum rubrum strain G-9, bacteriochlorophyll a was distributed in two bands after rate-zone sedimentation in sucrose density gradients. From the physicochemical properties of these fractions, it was concluded that the upper band consisted of small membrane fragments, whereas the major band was composed of fragmented vesicular intracytoplasmic membrane (chromatophores). After a pulse with L-[35S]methionine, apparent polypeptide subunits of the reaction center and light-harvesting complexes within the upper pigmented fraction were labeled more rapidly than those of chromatophores; after a chase with excess unlabeled L-methionine, radioactivity from these components within the upper band appeared to be chased into the corresponding polypeptides of chromatophores. These labeling patterns are interpreted to reflect growth initiation and maturation of the photosynthetic apparatus and may, in part, represent a general mechanism for the development of vesicular intracytoplasmic membranes.     

Kinetic Mechanisms of Biological Regulation in Photosynthetic Organisms

Principles of regulation on different levels of photosynthetic apparatus are discussed. Mathematical models of isolated photosynthetic reaction centers and general system of energy transduction in chloroplast are developed. A general approach to model these complex metabolic systems is suggested. Regulatory mechanisms in plant cell are correlated with the different patterns of fluorescence induction curve at different internal physiological states of the cells and external (environmental) conditions. Light regulation inside photosynthetic reaction centers, diffusion processes in thylakoid membrane, generation of transmembrane electrochemical potential, coupling with processes of CO₂ fixation in Calvin Cycle are considered as stages of control of energy transformation in chloroplasts in their connection with kinetic patterns of fluorescence induction curves and other spectrophotometric data.