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

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll alpha-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by less than 9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is condluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Radioactivity eventually destined for the chromatophore membrane of Rhodopseudomonas sphaeroides was shown in pulse-chase studies to appear first in a distinct pigmented fraction. This material formed an upper pigmented band which sedimented more slowly than chromatophores when cell-free extracts were subjected directly to rate-zone sedimentation on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified fraction contained polypeptide bands of the same mobility as light-harvesting bacteriochlorophyll a and reaction center-associated protein components of chromatophores; these were superimposed upon cytoplasmic membrane polypeptides. The pulse-chase relation was confined mainly to the polypeptide components of these pigment-protein complexes. It is suggested that the isolated fraction may be derived from sites at which new membrane invagination is initiated.     

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.     

Fusion of chromatophores derived from Rhodopseudomonas sphaeroides

Fusion of chromatophores, the photosynthetic membrane vesicles isolated from the intracytoplasmic membranes of Rhodopseudomonas sphaeroides, was achieved by the use of poly(ethylene glycol) 6000 as fusogen. Ultracentrifugation, electron microscopy, intrinsic density and isotope labeling were used to demonstrate chromatophore fusion. Although studies of the flash-induced shift in the carotenoid absorbance spectrum indicated that the membrane was rendered leaky to ions by either the fusion procedure or the increased size of the fused products, the orientation and integrity of fused chromatophores were otherwise demonstrated to be identical to control chromatophores by freeze-fracture electron microscopy, proteolytic enzyme digestion, enzymatic radioiodination, and transfer of chromatophore phospholipids mediated by phospholipid exchange protein extracted from Rps. sphaeroides.     

Membranes of Rhodopseudomonas sphaeroides VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromatophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (E_m = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

Membranes of Rhodopseudomonas sphaeroides. VI. Isolation of a fraction enriched in newly synthesized bacteriochlorophyll alpha-protein complexes.

Radioactivity eventually destined for the chromatophore membrane of Rhodopseudomonas sphaeroides was shown in pulse-chase studies to appear first in a distinct pigmented fraction. The material formed an upper pigmented band which sedimented more slowly than chromatophores when cell-free extracts were subjected directly to rate-zone sedimentation on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified fraction contained polypeptide bands of the same mobility as light-harvesting bacteriochlorophyll alpha and reaction center-associated protein components of chromatophores; these were superimposed upon cytoplasmic membrane polypeptides. The pulse-chase relation was confined mainly to the polypeptide components of these pigment-protein complexes. It is suggested that the isolated fraction may be derived from sites at which new membrane invagination is initiated.     

The functional unit of electrical events and phosphorylation in chromatophores from Rhodopseudomonas sphaeroides

1. From electron micrographs of chromatophores from Rhodopseudomonas sphaeroides and from the estimated bacteriochlorophyll content of the sample a mean value of 4700 bacteriochlorophyll per chromatophore was estimated. The mean diameter of the chromatophore vesicles was 600 Å.2. The decay of the flash-induced electric potential across the chromatophore membrane measured by the carotenoid band shift was 20% accelerated by about one valinomycin molecule per 4700 bacteriochlorophyll, i.e. by one ionophore molecule per chromatophore.3. The inhibition of the flash-induced ATP formation by valinomycin followed a similar pattern to the accelerated decay of the electric potential.4. The single turnover flash yield of ATP synthesis gave a mean value of one ATP per 1470 bacteriochlorophyll or about 3 ATP per vesicle.5. With regard to the partitioning of the ionophore between the membrane (85%) and aqueous phase (15%) we conclude that one molecule of valinomycin per chromatophore is sufficient to begin to collapse the electrical potential and inhibit ATP synthesis. It is therefore suggested that the membrane potential is an essential component of the energized state which is used for phosphorylation.The results correspond to those obtained for the 100-fold larger vesicles in chloroplasts (thylakoids) where one molecule of ionophore is also sufficient to quench both events.     

Structural and functional properties of chromatophores and membrane vesicles from Rhodepseudomonas sphaeroides

Membrane vesicles have been isolated by a modified procedure from Rhodopseudomonas sphaeroides, grown phototrophically under high light intensity. In addition,chromatophores have been isolated from this organism grown phototrophically with low light intensities.Structural, chemical and functional properties of both preparations have been investigated and compared. The orientation of the membrane preparations has been studied by freeze-etch electron microscopy, the localization of cytochrome c₂, and light-driven active transport of amino acids and Ca²⁺. The results demonstrate that the orientation of the vesicle membrane is the same as the cytoplasmic membrane of intact cells; the membranes in chromatophores, however, have an inverted orientation.On a dry weight basis, the membrane vesicles contain less protein, carotenoids and bacteriochlorophyll and more lipids than do chromatophores. Qualitatively, however, the composition of both preparations is similar.It is concluded that the intracytoplasmic structures from which the chromatophores are derived are structurally and functionally similar to (and most likely continuous with) the cytoplasmic membranes from which the vesicles are derived.     

Average distance between bacteriochlorophyll molecules on chromatophore membranes of Rhodopseudomonas sphaeroides

The average distance betweeen bacteriochlorophyll moleculeson the chromatophore membrane of Rhodopseudomonas sphaeroideswas estimated by two methods: measurement of the ratio of thenumber of chromatophores to the number of polystyrene-latexparticles mixed in a chromatophore suspension and measurementof the packed volume of chromatophores in the suspension. Theaverage distance was obtained under the assumption that bacteriochlorophyllmolecules are positioned at hexagonal lattice points. The valuewas 34 A{ring} when the bacteriochlorophyll molecules were assumedto be on one side of the membranes and 48 A{ring} when theywere assumed to be on both sides. (Received February 20, 1978; )     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature

Light-induced absorbance changes were measured at temperatures between ?30 and ?55 °C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the “primary” to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870⁺ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870⁺.A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artificial donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.     

THE PHOTOSYNTHETIC APPARATUS OF RHODOSPIRILLUM MOLISCHIANUM

By varying the light intensity and temperature during growth it is possible to obtain cultures of Rhodospirillum molischianum in which the specific bacteriochlorophyll contents differ by as much as fivefold. We used such cultures to compare the changes in the electron microscopic appearance of the cells with the changes in the amount and bacteriochlorophyll content of chromatophore material isolated from cell extracts. The cells contained a variable number of internal membranes which are invaginations of the cell membrane. The shape, size, number, and arrangement of the infoldings varied as the specific bacteriochlorophyll content of the cells changed. In cells with little bacteriochlorophyll, the invaginations were mostly tubular. In cells with larger amounts of bacteriochlorophyll, the invaginations were disc-shaped and the discs were appressed together in stacks of 2 to 10 discs each. Variations in the number of discs per stack could be accounted for by a simple statistical model. The average area per disc increased with increasing bacteriochlorophyll content. Quantitative estimations of the relative volumes occupied by membranes in cells with four different bacteriochlorophyll contents showed that the amount of internal membrane alone had no direct relationship with the bacteriochlorophyll content of the cells; however, the total amount of membrane (cell membrane plus internal membrane) was directly proportional to the bacteriochlorophyll content. The specific bacteriochlorophyll content of isolated chromatophore material was proportional to the bacteriochlorophyll content of whole cells; the total amount of chromatophore material was independent of the bacteriochlorophyll content of whole cells. Several possible explanations of this paradoxical discrepancy between the electron microscope observations and the analytical results are discussed.     

Differentiation of the membrane system in cells of Rhodopseudomonas capsulata after transition from chemotrophic to phototrophic growth conditions

Aerobically in the dark grown cultures of Rhodopseudomonas capsulata were shifted to low oxygen partial pressure for 30 min and afterwards to phototrophic conditions (anaerobic, light). During 210 min of adaptation to a phototrophic mode of life the bacteriochlorophyll (BChl) concentration increased 53-fold (doubling time 40 min) and the carotenoid content six fold. Growth was delayed. The light membrane fraction from chemotrophic and induced phototrophic cells contained low concentrations of small photosynthetic units (reaction center+light harvesting BChl B870), and low respiratory activities, especially of succinatecytochrome c oxidase. The heavy membrane fraction, i.e. the intracytoplasmic chromatophore fraction, increased during adaptation approximately 9-fold in surface area per cell, 42-fold in BChl content, 7-fold in reaction center content and 6-fold in the size of the photosynthetic unit.Phospholipid and fatty acid content and patterns changed slightly during adaptation.Non-standard Abbreviations BChl bacteriochlorphyll - R. Rhodopseudomonas     

Disappearence of the intracytoplasmic membranes inRhodopseudomonas sphaeroides

The photosynthetic bacterium,Rhodopseudomonas sphaeroides, can be grown phototrophically (light, anaerobiosis), of chemotrophically (dark, aerobiosis). In the first case, it contains intracytoplasmic membranes with photosynthetic pigments. When shifted from phototrophy to chemotrophy these membranes disappear in an unknown fashion. In the present experiment, samples were taken for electron microscopy, cell density and bacteriochlorophyll determinations after shift from phototrophy to chemotrophy. The density of intracytoplasmic vesicles was measured on micrographs. During the first 2h growth is very slow and the ultrastructure remains unaltered. As growth resumes, the vesicles disappear at a rate which implies that they are not incorportated into the cytoplasmic membrane, nor actively digested, but remain intact and become increasingly diluted in the cytoplasm as the culture grows. The size of the vesicles was estimated to about 500 Å. The number of vesicles in phototrophically grown cells was calculated to about 575 per cell, and after 6h chemotrophic growth to about 100. The areas of the cytoplasmic and intracytoplasmic membranes are roughly calculated.Abbreviations Bchl bacteriochlorophyll - CM cytoplasmic membranes - ICM intracytoplasmic membranes     

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides

A cell envelope fraction has been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm³) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm³) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O₂ pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm³). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.     

ATP-synthetase complex (F1F0) from Escherichia coli. Purification and characterization of subunits A and B of the F0 part

An intact B890 light-harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low-M_r polypeptide. Both these polypeptides are present in the intact chromatophore membrane. Analysis of the pigment content of this complex suggests that per pair of polypeptides the complex contains 2 molecules of bacteriochlorophyll and one molecule of carotenoid.     

Bacteriochlorophyll Synthesis and the Ultrastructure of Wild Type and Mutant Strains of Rhodopseudomonas spheroides

The ultrastructure of sectioned cells of mutant and wild type Rhodopseudomonas spheroides has been examined by electron microscopy. The characteristic vesicles associated with the presence of bacteriochlorophyll were found in wild type cells grown with low aeration. These were also found in mutant TA-R which forms bacteriochlorophyll under high aeration. None of the mutants with blocks in bacteriochlorophyll synthesis contained intracytoplasmic membrane. These included mutant 8-17 which accumulates bacteriochlorophyllide but fails at the phytolation step. We conclude that the intact bacteriochlorophyll molecule, or some particular membrane protein associated with it, is needed for the development of the characteristic intracytoplasmic membrane system in R. spheroides.     

Early formation of intracytoplasmic membranes in Rhodospirillum rubrum

Cytoplasmic and intracytoplasmic membranes were isolated from Rhodospirillum rubrum by equilibrium sucrose density gradient centrifugation. Immediately after the induction of photosynthetically active intracytoplasmic membranes, bacteriochlorophyll is incorporated predominantly into the cytoplasmic membrane. With increasing pigment concentrations the newly arising intracytoplasmic membranes become sites of preferential bacteriochlorophyll incorporation. During this process the infrared absorption band of the pigment shows a red shift. The shift is more pronounced with intracytoplasmic than with cytoplasmic membranes. Pulse-chase of cytoplasmic membrane proteins reveals that such proteins become constituents of intracytoplasmic membranes.     

Characterization of three membrane fractions isolated from cells of Rhodopseudomonas capsulata adapting from chemotrophic to phototrophic conditions

By means of sucrose density centrifugation three membrane fractions, named light, medium and heavy have been isolated from cells of Rhodopseudomonas capsulata strain 37b4, adapting from chemotrophic to phototrophic growth conditions. Succinate dehydrogenase activity of aerobically grown cells was mainly confined to the heavy (chromatophore) fraction. Upon changing to phototrophic conditions the activity of the succinate dehydrogenase increased in the medium and light fraction. All fractions contain bacteriochlorophyll. NADH dehydrogenase of chemotrophically grown cells was enriched in the light and medium fraction but is increased in the heavy fraction under phototrophic growth conditions. The capacity of photophosphorylation is high in the light and heavy fraction. The results indicate a differentially incorporation of functional subunits into specific parts of the membrane system during membrane differentiation.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N,N-dicyclohexyl carbodiimide     

Membranes of Rhodopseudomonas sphaeroides: effect of cerulenin on assembly of chromatophore membrane.

The effects of cerulenin were investigated in Rhodopseudomonas sphaeroides to elucidate further the mechanisms controlling the assembly of the chromatophore membrane. When this potent inhibitor of fatty acid biosynthesis was added to photosynthetically grown cultures, there was an immediate cessation of phospholipid, bacteriochlorophyll a, carotenoid, and ubiquinone formation. Concurrently, there was also a marked decrease in the rate of incorporation of protein into the chromatophore membrane. In contrast, only a small decrease in the rate of soluble and cell envelope protein synthesis was observed and, in chemotrophically grown cells, protein continued to be incorporated into both the cytoplasmic and outer membranes. The removal of delta-aminolaevulinate from mutant H-5 of R. sphaeroides, which requires this porphyrin precursor, was reexamined to determine whether cerulenin-induced cessation of chromatophore protein incorporation was due solely to blocked bacteriochlorophyll a synthesis. In the deprived H-5 cells, inhibition of [35S]methionine incorporation into chromatophores was confined mainly to apoproteins of bacteriochlorophyll a complexes. Other minor chromatophore proteins continued to be inserted to a greater extent than in cerulenin-treated wild type where phospholipid synthesis has also ceased. These results indicated that the assembly of the chromatophore membrane is under strict regulatory control involving concomitant phospholipid, pigment, and protein syntheses.     

Free-energy change accompanying the reduction of the reaction center secondary quinone in Rhodopseudomonas sphaeroides chromatophores

The standard free-energy change accompanying the electron transfer from Q_A to Q_B was estimated from the intensity of the delayed fluorescence in chromatophores of Rhodopseudomonas sphaeroides. The value of 120 meV (at pH 8) suggests that Q_B⁻ is more stable in the chromatophore membrane than in the isolated reaction center.