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We show in this paper that a complex constituted by phospholipids and LHI and LHII alpha polypeptides was inserted in a heavy membrane fraction in a nonextractable form, indicating a transmembrane localization. The best accepting membranes originated from aerobically grown cells. Addition of ATP during the insertion inhibited this reaction 25 to 30% in heavy membranes isolated from aerobically grown cells (HMaer) and a higher inhibition (60 to 65%) was detected when using heavy membranes isolated from photosynthetically grown cells (HMpho). Purification by gel filtration of a crude Na2CO3 extract yielded three phosphate-labeled fractions. Two of them contained protein and phospholipids in a stable association. However, only fractions containing phosphatidylethanolamine were shown to be reconstituted. The third radioactive fraction contained labeled ATP and protein, but no phospholipids and could not be reassociated to the heavy membranes of any origin. A model for the insertion of the LH polypeptides is presented in which the recently synthesized polypeptides are phosphorylated and become associated to anionic phospholipids. The interaction of this complex to the membrane spontaneously leads to stable insertion.

Mutants of Rhodobacter capsulatus, blocked at different steps of bacteriochlorophyll a (BChl) synthesis between protoporphyrin IX and 2-hydroxyethyl bacteriochlorophyllide a, were induced to synthesize the photosynthetic apparatus by lowering of oxygen tension in dark cultures. The cells were pulse-labeled with [35S]methionine and the radioactivity chased after dilution of [35S]. The specific radioactivity in the pigment-binding proteins of light-harvesting and reaction center proteins of the wild-type strain was not lowered during the chase period of three hours but in the BChl-free mutants the label disappeared within five to thirty minutes. The polypeptides were inserted into the membrane but did not remain stably incorporated. In the mutant strain NK9 the synthesis of the carotenoid spheroidenone/spheroidene was inhibited by insertion of Tn5 in the crtI gene (phytoene desaturase), which blocked completely the formation of the light-harvesting (LH) complex II (B800–850) but not of the LHI (B870) complex. In this mutant the polypeptides of the LH complexes were synthesized in a lower amount than in the wild-type cells and were inserted into the membrane. The LHIIα poly-peptide disappeared after 60 min of chase while the LHIIβ was more stable. It was concluded that the pigments are not only necessary for absorption of photons and efficient transfer of excitation energy but have a structural role by stabilizing the oligomeric LH complexes. This is in accordance with the crystal structure data of LHII complexes.

Membranes of Rhodobacter capsulatus strain U43 (pTX35) showed qualitatively very similar phosphorylation patterns under in vitro and in vivo conditions. In vitro, it was irrelevant whether the phosphate source was orthophosphate or ATP. Inhibitors of electron transport did not inhibit light-harvesting complex I (LHIα) (B870) polypeptide phosphorylation, except for o-phenanthroline, which was strongly inhibitory. Redox conditions regulated the amount of protein phosphorylated; external redox potentials between +200 and +300 mV promoted the reaction. Phosphorylation was inhibited by uncouplers such as carbonyl cyanide m-chlorophenyl hydrazone and nigericin plus valinomycin plus potassium ions. Inhibitors of the H+-ATPase were also inhibitory when the phosphate source was [32P]Pi or [γ-32P]ATP. From these results, it was concluded that an operative reaction center, a coupled membrane, and external redox potentials higher than +200 mV are required for optimum LHIα phosphorylation. We also demonstrated that phosphorylation of LHIα polypeptide occurs before insertion into the membrane and that phosphate is preferentially incorporated into specific domains within the cytoplasmic membrane. Intracytoplasmic membranes, identified here as light membranes, were found to contain a dephosphorylated LHIα polypeptide.

The spectral analysis of membrane fractions and a fraction enriched in light-harvesting (LH) complex I + reaction center (RC) of Roseococcus thiosulfatophilus, RB3 and of the membrane fraction and of the isolated LH II and LH I + RC complexes of Erythromicrobium ramosum, E5 is reported. Quantum yields of singlet energy transfer between carotenoids and bacteriochlorophyll (light-harvesting function) were calculated from comparison of absorption and fluorescence excitation spectra of the respective preparations. The results indicate that the excess of carotenoids in the membrane does not contribute to the lightharvesting function of the LH complexes.

Near-infrared excited Fourier-transform Raman spectra have been obtained from different spectral forms of Rhodobacter sulfidophilus light-harvesting II complexes. This complex, when isolated in lauryldimethylamine oxide, exists in a 805–828 nm form, which can be reversibly converted to the native 805–851 nm form upon addition of salt. The FT-Raman spectra predominantly show contributions of the carotenoid in the light-harvesting complex, with small but significant contributions of the bacteriochlorophylls excited in preresonance in the Q y transition. One strongly and one weakly interacting 2a acetyl C=O group as well as one moderately strong interacting and one non-interacting 9-keto C=O carbonyl modes of the bacteriochlorophylls can be discerned for the 805–828 nm form. Changes of relative band intensities caused by different resonance conditions for the different spectral forms lead to an assignment of the strongly interacting 2a acetyl C=O and the moderately strong interacting 9 keto C=O to bacteriochlorophylls organized in the 828 pigment moiety. Shifts of these bands to higher frequencies upon the salt-induced transition indicate a perturbation of the pigment-protein interaction, probably caused by a local protein conformational change.

Cells of Rhodobacter capsulatus wild-type strains (37b4, B 10) and mutant strains, lacking lightharvesting (LH) complex II (B800–850) and defective in formation of LH I (B870) complex [U 43 (pTXB 87), U43 (pTXA6-10)] were grown photosynthetically at high and low light intensities in a turbidostate. The mutant strain U43 (pTXA6-10), lacking any LH system, was able to grow at high and low light intensities with doubling times of 4.6 and 9.8 h, respectively. In this mutant the concentration of photochemical reaction centers (RC) per cell and per membrane protein was several times higher than in wild type cells, but the bacteriochlorophyll content, the size of the photosynthetic unit and the rate of photophosphorylation were lower than in wild type cells. Reversible bleaching of reaction center and photophosphorylation were measured under different excitation light intensities. The charge recombination in the RC between the primary donor and QB was very slow in the mutant strains. Two membrane fractions differing in absorption spectra and light saturation behaviour of reversible bleaching and photophosphorylation were isolated from the mutant strains. The experimental data indicate that photosynthetic units of different composition and/or organization are present in the mutant cells.

Photosynthesis was discovered by its metabolic aspect, the CO2 assimilation and the O2 production of green plants as a light-dependent process catalyzed by chlorophyll (J. Priestley 1772, J. Ingenhousz 1779, N. T. de Saussure, 1804, H. Dutrochet 1837). Justus Liebig demonstrated 1840 that plants can grow on pure inorganic nutrients (HCO3 -, NH4 +, SO4 2+ and trace elements). The law of maintenance of energy and the unit of heat (calorie) were discovered by J. Robert Mayer (1845), J. P. Joule (1818-1889) and Hermann L. Helmholtz (1821-1894), at the middle of the century, but the energetic aspect of photosynthesis, i.e. the transformation of light energy into chemical energy, was recognized many decades after demonstration of CO2 fixation. Wilhelm Engelmann wrote 1888 “The purple schizomycetes (bacteria) step in the group of organisms which assimilate like green plants. The bacteriopurpurin (bacteriochlorophyll) is a true chromophyll, so far the actual absorbed light energy is transformed in potential chemical energy”.

Roseobacter (Rs.) denitrificans belongs to the so-called aerobic photosynthetic bacteria. This type of bacteria is closely related to purple bacteria, but in contrast to these ‘true’ photosynthetic bacteria, they are not able to grow under anoxic conditions (1). Photosynthetic protein complexes and bacteriochlorophyll are only built under oxic conditions in these bacteria. Rs. denitrificans is phylogenetically close to Rhodobacter (Rb.) capsulatus a true phototrophic purple bacterium (2). The puf operon encoding the protein subunits L, M, C of the reaction center (RC) and of light harvesting complex I (LHI) from Rs. denitrificans were cloned and sequenced and show very high similarities to purple bacteria in either single sequences or the whole operon structure (3). Despite these similarities charge separation does not occur in the reaction center under anoxic conditions (4). The lack of photosynthetic activity was believed to be caused by the overreduction of the electron transfer system (1) and a higher midpoint redox potential of the primary acceptor (QA) of the RC in aerobic phototrophs compared to that of typical purple bacteria (5). For a better understanding of the charge separation in Rs. denitrificans RC the M and L subunits of RC from Rs. denitrificans were combined with the H-subunit from Rb. capsulatus in a Rb. capsulatus puf puc negative mutant (6). Charge recombination between the primary donor P3+ and Q A − was observed in the transconjugant indicating a correct assembly of L and M subunits but the transconjugant did not grow under anoxic conditions. In Rhodobacter sphaeroides it was shown that the H-subunit has an influence on electron transfer from QA to QB (7, 8). Since the H-subunit could be responsible for the differences between purple bacteria and aerobic phototrophs we cloned and sequenced the RC H-subunit from Rs. denitrificans. To get informations about the interaction between the quinones and the surrounding protein matrix a model of the HLM-complex of the RC was calculated.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.

The light-harvesting (LH) complex I (B870) of anoxygenic photosynthetic purple bacteria is the oligomeric form of its subunit B820 consisting of the low-molecular-weight polypeptides alpha, beta, bacteriochlorophyll (BChl), and carotenoids in the stoichiometric ratio [alpha1 beta1 (BChl2) Crt1-2]n. LHI surrounds the photochemical reaction center (RC). The major absorption band of the LHI complex is species-specific and is found at 870-890 nm; those of the subunit and the monomeric BChl a (dissolved in methanol) absorb at 820 and 770 nm, respectively. The isolated LHI complex can be reversibly dissociated to the B820 subunit or to the polypeptides and pigments by addition of detergents. Reconstitution of the B820 or the functional B870 complex is still possible after partial truncation of the N- or C-terminal regions of the alpha- or beta-polypeptide or of the beta-polypeptide only. The minimal structural requirements for reconstitution of a spectrally wild-type form after truncation of the polypeptides and/or modifications of the BChl molecule are described. The insertion of the LHIalpha- and LHIbeta-polypeptides into the membrane and the in vivo assembly of LHI, studied in a cell-free system and in whole cells of Rhodobacter capsulatus, depend on the primary structures of both polypeptides, BChl, the chaperones DnaK and GroEL, membrane-bound proteins, and energized membranes. Exchanges, deletions, or insertions of amino acyl residues, especially in the conserved region of the N-terminus of the LHIalpha-polypeptide, prevent or reduce the efficiency and stability of the LHI assembly. Therefore, reconstitution of LHI in a detergent micelle does not exactly reproduce the formation of the LHI complex in the photosynthetic membrane in vivo. The N-terminal domains play a crucial role in the formation of the oligomeric protein scaffold and of the pigment array. Facultatively phototrophic bacteria such as Rhodospirillum (Rsp.) rubrum or Rhodobacter (Rba.) capsulatus can adjust to changes in oxygen tension, light intensity, temperature, and substrates to grow under chemotrophic or phototrophic conditions. The photosynthetic apparatus (PSA), localized mainly on intracytoplasmic membranes (ICM), is usually synthesized only under low oxygen partial pressure. The cellular amount and composition of the PSA are modified upon changing light intensity in relation to cell growth (Drews and Golecki 1995). The morphogenesis of cellular structures like ICM is quite different from self-assembly. Self-assembly is a reversible process of aggregation of the constituents of a complex structure without protein synthesis and is driven by weak or strong forces in the interactions of the constituents. Morphogenesis results from the interplay of numerous gene products and the cellular organization and is always dependent upon pre-existent structures (Harold 1995). The morphogenesis of the photosynthetic membrane in purple bacteria has been studied in its different steps. The regulation at the transcriptional and post-transcriptional levels in purple bacteria, and the structure and morphogenesis of the ICM have been described recently (Armstrong 1995; Bauer 1995; Biel 1995; Drews and Golecki 1995; Klug 1995). In this mini-review, I will focus on the minimal requirements for the in vitro assembly of light-harvesting (LH) complex I (B870) from its constituents in detergent micelles and compare the results with observations on the complex process of targeting and import of LHI polypeptides into the membrane and assembly of B870.

A respiring culture of Rhodobacter sphaeroides, grown in the dark under defined aerobic conditions, produced cells capable of immediately commencing adaptation to photosynthetic growth on exposure to light and further reduction of oxygen tension. Adaptation was complete after 12 h and the bacteriochlorophyll a content increased 10–20-fold. This adaptation was performed in the presence of either H218O or 18O2. The extracted bacteriochlorophyll a was examined by mass spectrometry to determine the origin of both the 3-acetyl and 131-oxo oxygen atoms: both were derived from water. The derivation of the 131-oxo group from water in R. sphaeroides indicates that the formation of isocyclic ring E from the 13-propionic acid methylester side chain of Mg2+-protoporphyrin IX mono-methylester is an anaerobic process involving a hydratase. This is very different to the situation in higher plants and green algae where the formation of isocyclic ring E is an aerobic process in which the 131-oxo group is derived from molecular oxygen via an oxygenase. In contrast to adapting R. sphaeroides cells, the 131-oxo group of bacteriochlorophyll a in growing cells of the obligate aerobic chemotrophic bacterium Roseobacter denitrificans, was labelled by 18O2 and is, therefore, derived from molecular oxygen like in higher plants and green algae; however, the 3-acetyl group was not labelled by 18O2. Thus, while the 131-oxo group has different origins in R. sphaeroides and R. denitrificans, the 3-acetyl group arises in both bacteria by enzymic hydration of the vinyl group of a chlorophyll a derivative.

Transcripts of the genes pufBA, pufB or pufA from Rhodobacter capsulatus were translated in a cell-free system of R. capsulatus. The incorporation of the nascent polypeptides LHIα and β in various types of membranes and the assembly of the light-harvesting (LH) complex I (B870) were investigated. The highest rate of stable incorporation of LHIα and β into the membrane was observed with membranes from the wild type strain grown under chemotrophic conditions. Addition of membranes from cells defective in biosynthesis of pigment-binding proteins resulted in a less efficient or less stable incorporation of LHIαβ. The single polypeptides LHIα or β were synthesized and inserted into the membrane but were extractable to a higher percentage by 6 M urea than the pairwise inserted LHI polypeptides. If the ribosomes and the S135 extract were depleted of DnaK the rate of synthesis of both polypeptides, LHIα and β, was strongly reduced. Removal of GroEL from the cell-free system did not impair the synthesis and membrane association of both proteins, but affected the stable insertion. A high percentage of the LHIαβ polypeptides became extractable by 6 M urea if the cell-free system was depleted of GroEL. Addition of GroEL to the cell-free system restored the capacity of stable insertion of both proteins into the membrane. GroEL interacted with LHIα and β before membrane targeting as shown by immunological means. A protein fraction, which can be removed from the membrane with a low-salt buffer, supported the effective and stable incorporation of LHIαβ into the membrane. It is concluded that the assembly of the LHI complex in the membrane system of R. capsulatus is a multistep process guided and supported by polypeptides located in the cytoplasm and in the membrane. In the cell-free in vitro system not only the correct insertion of the LHI polypeptides but also an assembly with bacteriochlorophyll was observed. BChl was synthesized from δ-amino levulinate in the cell free system.

The photosynthetic bacterium Rhodobacter sulfidophilus is able to grow chemotrophically and phototrophically at a broad range of light intensities. In contrast to other facultative phototrophs, R. sulfidophilus synthesizes reaction center and light-harvesting (LH) complexes, B870 (LHI) and B800-850 (LHII) even under full aerobic conditions in the dark. The content of bacteriochlorophyll (BChl) varied from 3.8 micrograms Bchl per mg cell protein when grown at high light intensity (20,000 lux) to 60 micrograms Bchl per mg cell protein when grown at low light intensities (6 lux). After a shift from high light to low light conditions, the size of the photosynthetic unit increased by a factor of 4. Chromatographic analysis of the LHII complex, isolated and purified from cells grown phototrophically (at high and low light intensities) and chemotrophically, could resolve only one type of alpha and one type of beta polypeptide in the purified complex, of which the N-terminal sequences have been determined.

We analyzed the 16S ribosomal DNAs of three obligately aerobic, bacteriochlorophyll a-containing bacteria, "Roseococcus thiosulfatophilus," "Erythromicrobium ramosum," and new isolate T4T (T = type strain), which was obtained from a marine cyanobacterial mat. "Roseococcus thiosulfatophilus" is a member of the alpha-1 subclass of the Proteobacteria and is moderately related to Rhodopila globiformis, Thiobacillus acidophilus, and Acidiphilium cryptum (level of sequence similarity, 90%). "Erythromicrobium ramosum" and isolate T4T are closely related to Erythrobacter longus and Porphyrobacter neustonensis (level of sequence similarity, 95%). These organisms are members of the alpha-4 subclass of the Proteobacteria. Strain T4T is a motile, red or orange bacterium. The major carotenoids are bacteriorubixanthinal and erythroxanthin sulfate. In vivo measurements revealed bacteriochlorophyll absorption maxima at 377, 590, 800, and 868 nm. Strain T4T grows in the presence of 5 to 96/1000 salinity and uses glucose, fructose, acetate, pyruvate, glutamate, succinate, and lactate as substrates. On the basis of its distinct phylogenetic position and phenotypic characteristics which are different from those of Erythrobacter longus, we propose that strain T4T should be placed in a new species of the genus Erythrobacter, Erythrobacter litoralis. The descriptions of "Roseococcus thiosulfatophilus" and "Erythromicrobium ramosum" are emended.

The obligate aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 contain numerous polar carotenoids. The major carotenoid of the strain RB3 was the C30 carotene-dioate (4,4′-diapocarotene-4,4′-dioate) and the respective diglycosyl ester which have never been isolated before from a bacteriochlorophyll containing bacterium. Strain E5 contains the very polar erythroxanthin sulphate. The major carotenoid bound to reaction center and light-harvesting complexes is bacteriorubixanthinal. Most of the carotenoids of both strains are not bound to the pigment-protein complexes of the photosynthetic apparatus but to the envelope fraction (cytoplasmic membrane and cell wall).

Reaction center (RC) and light-harvesting (LH) or antenna complexes are the major pigment-proteins of the photosynthetic apparatus of non sulfur purple bacteria. They are localized on the intracytoplasmic membranes. The LH-complexes serve to gather light-energy and funnel it to the photochemical RC where the excitation energy is transduced into a charge separation state and a redox potential difference. Reaction centers are surrounded by a constant number of core antenna complexes (8870 or B1020). Most species have a second and variable light-harvesting (LH) complex (B800–850) which interconnects the core complexes (Drews 1985). The LH complexes are oligomers of basic subunits which consist of two different small pigment-binding polypeptides α and β, having Mr of about 5000 to 7000. These polypeptides are amphiphilic proteins and span the membrane only once by a central hydrophobic domain (Fig. 1). The N- and C-terminal domains consist of polar, charged and hydrophobic amino acid residues and are exposed on the membrane surface. Two or three bacteriochlorophyll (Bchl) and one or two carotenoid molecules are bound non-covalently to the α and β polypeptides.

The B800–850 light harvesting complex of Rps. acidophila and the RC—B875 reaction center complex of Rps. palustris were crystallized in presence of detergents. The crystals were obtained by vapour diffusion technique using polyethylene glycol 1000 (1,2). For spectroscopic studies, the B800–850 and the RC—B875 complexes were crystallized on thin cover slides, allowing the crystals to grow to sufficient size in two dimensions. Sealed by a second cover slide, the crystals were mounted on a goniometer sample holder in a micro-spectrophotometer (3) built in our laboratory.

The photosynthetic apparatus of Rhodopseudomonas palustris contains, in addition to reaction center bacteriochlorophyll (Bchl) two spectral forms of light harvesting (LH) Bchl, i.e. LH Bchl I, characterized by an infrared absorption maximum at 880 nm (890 nm at 77 degrees K) and LH Bchl II absorbing at 805 and 855 nm (805 and 870 nm at 77 degrees K). LH Bchl I seems to be associated with a single protein species of an apparent mol. wt. of 13 000 whereas LH Bchl II is apparently associated with two proteins of mol. wts. of 9000 and 11 000. Cells in anaerobic cultures adapt to changes of light intensity 1. by variation of the size of the photosynthetic unit, i.e. the molar ratio of LH Bchl II to reaction center Bchl, 2. by variation of the number of photosynthetic units per unit of membrane area, 3. by regulation of the size of the intracytoplasmic membrane system. During adaptation of changes of oxygen partial pressure cells are able to synthesize reaction center Bchl, LH Bchl and intracytoplasmic membranes at different rates. The synthesis of reaction center Bchl and LH Bchl I are, however, coordinated with each other, while the synthesis of LH Bchl II and reaction center Bchl proceed independently.

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg ( p O 2 ). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll. Cells which were incubated at 0.5 mmHg ( p O 2 ) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of 14 C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.

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.

Lipopolysaccharides have been isolated from eight strains of the unicellular cyanobacterium Synechococcus. Fucose, mannose, galactose, glucose and glucosamine were found in all of the lipopolysaccharides investigated. Additionally, strain-specific sugars are present and permit the chemotyping of lipopolysaccharide. Chemotype I, comprising three strains with a high G+C content of DNA (71-66 mol%), is characterized by a high rhamnose portion and by 3,6-dideoxy-d-arabino-hexose (tyvelose). Chemotype III, represented by three strains with a low G+C content of DNA (55-48 mol%), contains a mannose-polymer with small amounts of 3-O-methyl-mannose, 4-O-methyl-mannose, 2-keto-3-deoxyoctonate and mannosamine. Lipopolysaccharides of the two strains of chemotype II contain 2,3,4-tri-O-methyl-arabinose. Lipid A is difficult to split off from the polysaccharide moiety, but is present in all lipopolysaccharides from the Synechococcus strains. The presence of Lipid A is supported by the finding of β-hydroxy fatty acids, predominantly β-hydroxypalmitic acid. The distribution of branched β-hydroxy fatty acids, detected in small amounts, parallels chemotyping of lipopolysaccharide based on the sugar composition. The phosphorus content of the lipopolysaccharides is low. The pyrogenicity of lipopolysaccharides from two strains is low. Synechococcus lipopolysaccharides have little reactivity in antisera raised in rabbits against homologous cells. As far as tested they do not migrate in immunoelectrophoresis. This confirms the neutral character or low negative charge of Synechococcus lipopolysaccharides.

Strain-specific low-molecular-weight polysaccharides of different chemical compositions were obtained from cells of nine different wild-type strains of the phototrophic bacterium Rhodopseudomonas gelatinosa. The polysaccharides are free of typical capsule components like hexuronic or aminohexuronic acids but contain (except that of strain 39/2) substantial amounts of phosphorus. A number of unusual o-methyl sugars (2-o-methyl-D-galactose, 2,3-di-o-methyl-D-galactose, 2-o-methyl-L-fucose) as well as 3,6-dideoxy-D-xylo-hexose (abequose) were identified in the R. gelatinosa polysaccharides. o-Methyl and dideoxy sugars however, are typical constituents of O-specific chains of the lipopolysaccharides of gram-negative bacteria (Rhodospirillaceae and Enterobacteriaceae, respectively). Considering both the R-type character of the R. gelatinosa lipopolysaccharides and the occurrence of these strain-specific ETEROPOLYSACCHARIDES, THE ASSUMPTION SEEMS TO BE JUSTIFIED THAT THE LOW-MOLECULAR-WEIGHT POLYSACCHARIDES ARE RELATED TO O-specific chains of lipopolysaccharides (haptens) rather than to capsular or slime antigens. In serological terms the polysaccharides of R. gelatinosa have to be classified as K-antigens. They are able to cover the O-specificity of the respective different strains and confer on them additional specificity which is demonstrable by bacterial agglutination.

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.

SUMMARY: A high molecular weight polysaccharide sedimenting at 105 000 g was extracted into the water phase of phenol/water extracts of Chlorogloeopsis PCC 6912 cells. The main sugars of the heteropolysaccharide were mannose, 6-O-methyl-d-mannose, glucose, rhamnose, and glucuronic and galacturonic acids. 6-O-Methyl-d-mannose was present partly in terminal linkage and partly 1,3-chain-linked, as revealed by methylation analysis. 3-O-Methyl-mannose, however, was found in only trace amounts and exclusively occupied terminal positions. The heteropolysaccharide presumably represented a polysaccharide of the cell wall. There was no indication of a lipid moiety. Application of procedures commonly used for lipopolysaccharide extraction from whole cells resulted in very low yields or even indicated complete lack of a typical lipopolysaccharide.

The near infrared absorption spectra and their fourth derivative were measured in membrane preparations or in the isolated light harvesting pigment-protein complex B 800-850 from Rhodopseudomonas capsulata in order to know how the underlying molecular absorption spectra contribute to the observed absorption curve. In contrast to the observations of Cogdell and Crofts [Biochim. Biophys. Acta 502, 409 (1978)] no splitting of the 855 nm absorption bands was observed at 300 K, 100 K, and 4 K. However, a small but significant splitting of the 870 nm band in all derivates (2nd, 4th, 6th) was observed at 300 K and 4 K. The lack of splitting of the 855 nm absorption band will be discussed in the light of the molecular organization of the B 855 moiety of this light harvesting bacteriochlorophyll-protein complex.

The O-antigen (lipopolysaccharide) of Anacystis nidulans, strain KM, has been isolated from whole cells and from cell wall preparations by phenolwater extraction. The polysaccharide moiety consists of a D-mannose polymer accompanied by smaller amounts of 3- and 4-O-methyl-D-mannoses, D-galactose, D-glucose, L-fucose, D-glucosamine, mannosamine and 2-keto-3-deoxyoctonate. Aldoheptoses are lacking. The degraded polysaccharide is split from lipid A by acid hydrolysis (10% acetic acid, 100°C, 3 h) whereby 2-keto-3-deoxyoctonate is released in small amounts. Degraded polysaccharide forms only one major fraction by Sephadex G-50 gel-filtration. This fraction includes all the sugars mentioned above except L-fucose, which is released during the acetic acid degradation. Periodate studies and methylation analysis revealed that the poly-mannose chain consists of about 75% 1→3 linked and of 25% 1→4 linked D-mannose units. Lipid A of A. nidulans is phosphate-free. The main fatty acid, β-hydroxypalmitic acid, is exclusively amide-bound, presumably to the amino group of D-glucosamine. Other fatty acids, found as minor constituents, are β-hydroxymyristic, palmitic and stearic acids. Lipopolysaccharide of A. nidulans KM exhibits high anticomplementary activity in guineapig serum. It is about 800 times less toxic for adrenalectomized mice than endotoxin from Salmonella typhimurium. The isolated lipopolysaccharide reacts with rabbit antisera against living or heat-killed cells of A. nidulans in passive hemagglutination, when untreated or heated, but not when alkali-treated lipopolysaccharide is used for red blood cell sensibilization. It is concluded that lipopolysaccharide of A. nidulans KM is exposed on the surface of the cell.

Formation of the photosynthetic apparatus was induced in aerobically grown dark cultures of Rhodopseudomonas capsulata by lowering of the oxygen tension. Besides the wild type strain the carotenoid-less mutant strain A1a+ was investigated. Both strains exhibited initially a decrease of the molar ratio of total bacteriochlorophyll (Bchl) to reaction center (RC) Bchl, followed by an increase. Synthesis of RC-Bchl preceded the synthesis of light-harvesting (LH) Bchl. Activities of photophosphorylation in membrane preparations, isolated from cultures after different periods of incubation at low aeration, decreased on the basis of total Bchl from about 9 to 2 mumole ATP/mumole total Bchl-min, whereas the rate on the basis of RC-Bchl remained constant (about 500 mumole ATP/mumole RC-Bchl-min). Under the same conditions the membrane proteins were labelled with U-14C-protein hydrolysate. Corresponding to RC-Bchl the synthesis of RC-proteins dominated during the first 30 min of incubation at PO2 below 3 mm Hg. After 45-60 min of membrane formation at low aeration the synthesis of LH-complex proteins exceeded the synthesis of RC proteins. The correlations between protein and Bchl synthesis in the sequential formation of RC- and LH-complexes are discussed.

Lipopolysaccharides (LPS, O-antigens) of 12 strains of the photosynthetic bacterium Rhodopseudomonas gelatinosa were obtained by the phenol/chloroform/petroleum ether method, recommended for extracting lipophilic glycolipids of enterobacterial R-mutants. All R. gelatinosa LPS have essentially the same chemical composition. Similar to LPS of Salmonella R-mutants of chemotypes Rd1 and Rd2, the sole neutral sugar constituent is an aldoheptose. The heptose of R. gelatinosa LPS has the D-glycero-D-manno- configuration, in contrast to the L-glycero-D-mannoheptose of enterobacterial LPS. 2-Keto-3-deoxyoctonate forms the acid-labile linkage between the lipid moiety (lipid A) and the oligosaccharide moiety of R. gelatinosa LPS. Like enterobacterial lipid A, lipid A of this species contains phosphate and D-glucosamine as the sole amino sugar. The fatty acid spectrum conprises beta-hydroxycapric, lauric, and myristic acids. Beta-Hydroxymyristic acid, the typical fatty acid of enterobacterial LPS, is lacking. The R. gelatinosa LPS show O-antigenic acitivity; passive hemagglutinations with untreated or heat-treated (not well alkali-treated) LPS and antisera prepared against heat-killed cells yield high titers. According to the serological cross-reactions observed, the LPS of the 12 strains could be arranged into two different serotypes: serotype I comprising strains 29/1, 29/2, 25/2, and serotype II comprising strains 44/K/6, 3/1, IS/10, 39/2, Dr2, 2150, P8P9, K32, P18f3.1. No serological cross-reactions were observed between LPS of these two different serotypes in passive hemagglutinations.

The light-harvesting antenna complex II of Rhodopseudomonas capsulata is characterized by two prominent IR-absorption maxima at 802 and 855 nm and three polypeptides of 14,000, 10,000 and 8,000 molecular weight (SDS-polyacrylamide gel electrophoresis). Bacteriochlorophyll (Bchl) is associated with the two lower molecular weight polypeptides. The membranes of the mutant Y 5, which lades reaction center and light harvesting complex I (B 875), were treated with trypsin. The 14,000 polypeptide was rapidly digested by trypsin, but the absorption spectrum of the membrane and the activity of the cytochrome c oxidase were not altered. Subsequently the 8,000 polypeptide was degraded. The digestion of the 8,000 polypeptide was concomitant with and proportional to the loss of absorbance at 802 nm. The absorption peak at 855 nm and the content of the 10,000 molecular weight polypeptide were, in contrast, stable for a longer time, but were also lost simultaneously. These results, in combination with the recent publications of Sauer and Austin (Biochemistry 17, 2011, 1978), and Cogdell and Crofts (Biochim. Biophys. Acta 502, 409, 1978) support the idea that the two molecules of Bchl associated with the 10,000 polypeptide are responsible for the 855 nm peak while the one or two mol of Bchl associated with 8,000 polypeptide result in the 802 nm absorption maximum.

The photosynthetically negative mutant strain Y142 of Rhodopseudomonas capsulata, which synthesizes bacteriochlorophyll (Bchl), carotenoids and the light-harvesting (LH) complex B800–850, but no reaction center and LH complex B870, is capable of synthesizing the Bchl-binding polypeptide (α, 12 kDa) of B870. In contrast to the high stability of the polypeptides of the B800–850 complex, the 12 kDa polypeptide was rapidly degraded after synthesis and insertion into the membrane.

Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 H + e − with uptake of 1 H + e − from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K+ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c2-oxidoreductase showed a pumping activity of 0.9 H + e − , which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.

Three plasmids containing the transposon Tn5, i.e. pSUP201::Tn5, pACYC184::Tn5 and pJB4JI were transferred from Escherichia coli to Rhodopseudomonas capsulata in order to mutagenize the genome. Mutants defective in bacteriochlorophyll and carotenoid synthesis and mutants unable to form the photochemical reaction center or one of the light-harvesting complexes were isolated. Of special interest were mutants that could not form the light-harvesting complex B800-850. Two of these mutants synthesized only two of the three polypeptides of this complex whereas the corresponding near infrared absorbance bands were not observed. Complementation analysis with the Rprime plasmid pRPS404, which contains a 50 kb region of the genome of R. capsulata carrying most genes responsible for expression of photosynthetic apparatus, revealed that some genes of the B800-850 light-harvesting complex lie outside this photosynthetic gene cluster.

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala+) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala+ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.

A new procedure for isolation, purification and identification of the three polypeptides of the membranebound light-harvesting complex I1 (BSOO-850) of Rhodopseudomonas capsulata has been developed. The polypeptides were extracted from crude intracytoplasmic membranes with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60. The peak fractions were transferred to solvents of different polarity and separated by gel filtration or ion-exchange chromatography. The three major polypeptides isolated by this two-step chromatography were found to be homogenous and identical with the three polypeptides of the light-harvesting complex 11, as judged by amino acid analysis and N-terminal sequence determination. Contaminating minor polypeptides, of which the functions are unknown, were different from the polypeptides of the B800-850 complex studied by the same criteria. The photosynthetic apparatus of Rhodopseudomonas capsuluta contains three bacteriochlorophyll-carotenoid-protein complexes, the photochemical reaction center and the two light-harvesting complexes I (B870) and I1 (BSOO-850). These complexes have been solubilized by detergent treatment from the membrane and purified [I, 21. The light-harvesting complex I1 contains three polypeptides. The smaller polypeptides are stoichiometrically associated with bacteriochlorophyll a and carotenoids forming the pigment moieties absorbing at 800 nm and 855 nm respectively [2,3]. The native B800-850 complex seems to consist of a tetramer of the basic unit, having an apparent M, of 180000 [4]. The three polypeptides have been isolated from the complex and characterized by amino acid composition and N-terminal sequence [2]. The isolation procedure was time-consuming and resulted in a low yield. In order to obtain highly purified polypeptides in larger amounts simple and efficient methods were developed. The polypeptides were extracted directly from freeze-dried crude membranes and purified to homogeneity in two chromatographic steps. The relevance of this procedure for isolation of integral membrane proteins and some properties of the isolated polypeptides are discussed.

The mutant strains A l a+ and Y 5 of Rhodopseudomonas capsulata are defective in formation of the light-harvesting complexes B800-850 and B870, respectively, but synthesize the large bacteriochlorophyll-binding polypeptides of the respectively complexes, shown by amino acid composition, N- or C-terminal sequence and immunoblotting. The stable assembly of the light-harvesting complexes in the membrane seems to be dependent from the coordinated synthesis of all pigment and protein components.

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c′ into the medium and lack cytochrome c 2.

The small bacteriochlorophyll-binding polypeptide of the light-harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C-terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.

The synthesis of bacteriochlorophyll (Bchl) was inhibited in wild type strain of Rhodopseudomonas capsulata by levulinic acid (LA), δ-aminolevulinic acid of α,α′-dipyridyl. The transcription of genes for pigment-binding proteins, the synthesis and incorporation of these proteins into the membrane and the assembly of photosynthetic complexes were studied. Inhibition of Bchl synthesis did not impair formation of mRNA for pigment-binding polypeptides, but inhibited the synthesis of these proteins as well as stable incorporation into the membrane. The results let suggest, that Bchl is necessary for the stabilization of pigment-binding proteins in the membrane, and that intermediates of Bchl-synthesis affect the synthesis of these proteins. The same experiments were carried out with mutant strains blocked in Bchl synthesis. All Bchl-less mutant strains investigated here, showed transcription of photosynthetic genes at a rate which was independent of oxygen tension. The synthesis of polypeptides of reaction center, B870 and B800-850 complexes were differently affected by blocks in Bchl-synthesis. LA inhibited the synthesis of pigment-binding proteins in mutant strains similarly as in wild-type strain.

Photophosphorylation activity under saturating continuous illumination was estimated in differently treated chromatophores of a B800–850 less mutant of Rhodopseudomonas capsulata . The activity was decreased when the chromatophores were fused with liposomes or when inhibitors of the H + ATPase or electron transport were added. Estimation of the irradiance needed for half-maximal turnover (apparent K m for light) revealed that fused membranes and inhibitor-treated chromatophores behaved oppositely. In fused membranes the K m value was increased 5-fold compared to the control chromatophores, from which we conclude that part of the input energy is dissipated and lost for photophosphorylation. Partial inhibition of H + ATPases or especially of electron transport resulted in a considerable decrease in the K m value. To explain this, we define a photosynthetic chain as the minimum functional unit being able to convert light energy into ATP. The decrease of the K m value indicates that photosynthetic chains are not separated from each other in the membrane and exchange at least one of the intermediates, ubiquinone, cytochrome c , or protons.

SUMMARY: A sheath fraction was isolated from cells of the cyanobacterium Chlorogloeopsis PCC 6912. It had a fibrillar fine structure, a high density and contained 38% (w/w) carbohydrates and 22% (w/w) protein. Glucose, mannose, galactose, arabinose, xylose, an unknown sugar and glucuronic acid were the major carbohydrates. Lipids were almost entirely absent. The protein was not solubilized by treatment with hot phenol/water (68 °C; 20 min) or Triton X-100 (2%, w/v; 20 °C; 3 h). The carbohydrates of the sheath belong to two different polysaccharides. The major one was not separated from the protein moiety of the sheath by hot phenol/water. It contained glucose as the main constituent, arabinose and xylose but little galactose, in addition to the other carbohydrates. In contrast, the minor polysaccharide was soluble in the water phase of hot phenol/water extracts and contained galactose as a major constituent in addition to glucose and mannose, but very little arabinose and xylose. The polysaccharide enriched in galactose could also be obtained from the water phase by precipitation with Cetavlon when sheath-containing whole cells were extracted by phenol/water.

Photooxidation of endogenous cytochrome(s) c, photoreduction of endogenous chrome(s) b and photobleaching of bacteriochlorophyll have been demonstrated in an in vitro reconstituted system, previously demonstrated to support photophosphorylation. The kinetic responses of these redox reactions to substrate and antimycin A in these particle are characteristic of electron transport processes and stronglysupport the contention that all, or a part of, the oxidative phosphorylation electron transport pathway can be coupled to reaction center, photopigment complex in a manner which supports photophosphorylation. In addition, a succinate-supported light dependent reduction of NAD+ was found.

The small bacteriochlorophyll-binding polypeptide(β) of the light-harvesting complex B800-850 from the phototrophic bacterium Rhodopseudomonas capsulata was purified from the complex and the membrane, respectively. The complete amino-acid sequence has been determined. The M r of the polypeptide is 4597. The protein consists of 49 amino acids, 64% of which are hydrophobic. Homology with other bacteriochlorophyll-binding polypeptides is discussed. Bacteriochlorophyll-binding polypeptide Primary structure Rhodopseudomonas capsulata B800-850 antenna pigment complex

Lowering of oxygen tension in dark cultures of Rhodobacter capsulatus wild type induces formation of three pigment-protein complexes. The mutant strain GK2 formed the B800–850 antenna complex only when grown anaerobically in the light but not in the dark. Under phototrophic culture conditions bacteriochlorophyll, bacteriopheophorbid, and B800–850 polypeptides were synthesized in larger amounts than in chemotrophically grown cultures. The B800–850 complex of the mutant strain GK2 was formed in the absence of carotenoids. Messenger RNAs for reaction center B870 and B800–850 proteins were synthesized in chemotrophic and phototrophic cells. The formation of mRNA in GK2 differed quantitatively from the expression in the wild type strain.

The photosynthetic apparatus of the facultative phototrophic bacterium Rhodopseudomonas capsulata contains three bacteriochlorophyll-carotenoid-protein complexes: the reaction center and the light-harvesting (LH) antenna complexes LHI (B870) and LHII (B800--850). In contrast to green anoxygenic phototrophic bacteria and the oxygenic cyanobacteria, the light-harvesting complexes of Rhodospirillaceae and Chromatiaceae are integral membrane particles. Variations in light fluxes induce membrane differentiation mainly expressed as variations in the size of the photosynthetic unit and in the area of intracytoplasmic membrane per cell. The B800--850 complex is the variable part of the photosynthetic apparatus. Synthesis of bacteriochlorophyll and of the polypeptides of the pigment complexes was found to be strongly coordinated. The synthesis of these polypeptides was followed immediately by the assembly of the complexes in the membrane. Bacteriochlorophyll or a signal substance triggered by bacteriochlorophyll synthesis regulated the synthesis of these polypeptides at the level of translation. The pigment-binding subunits of the B800--850 complex form oligomeric structures which interact with subunit H of the reaction center. A model of the topographical relationships of the pigment complexes is discussed.

Intracytoplasmic membranes of wild type strain 37 b 4 and mutant strains A1a car-bchl-, A1a car-bchl+ ofRhodopseudomonas capsulata were isolated. The membrane proteins were solubilized and separated by polyacrylamide gel electrophoresis (methods of Takayamaet al., 1964; Weber and Osborn, 1969). The band patterns were compared with each other. From the strain A1a car-bchl+ reaction center particles were isolated by treatment of membrane with Triton X-100 followed by sucrose density gradient centrifugation. The reaction center particles were found to be enriched in reaction center bacteriochlorophyll. This pigment shows a reversible bleaching at 855 nm and a blue shift at 798 nm. The light harvesting bacteriochlorophyll portion of this fraction was 14–22% of the total bacteriochlorophyll content. The three main proteins of the reaction center particles amount to about 80% of the total protein of the particles. The molecular weights of the main proteins were estimated to be 32000, 27500 and 22500 daltons.

1. 1. Two pigmented bands from cell-free extracts of light-growth Rhodospirillum rubrum could be isolated in a linear gradient of Ficoll by zone centrifugation. 2. 2. As shown by several methods, the lower band of the gradient contained chromatophores (thylakoids) in a highly purified form. 3. 3. In the upper band of the gradient, particles with an average diameter of 360 A could be identified by electron microscopy. 4. 4. Electrophoresis of the contents of the upper band in agar gel (0.5%) made it possible to separate two zones. Whereas only protein could be determined in the faster migrating zone, protein, pigments, lipids and activity of succinate dehydrogenase could be determined in the slower migrating zone. 5. 5. The chromatophores (thylakoids) of the lower band were split by treatment with phenol-formic acid-water (2:1:1, v/v/v) and then separated by gel electrophoresis in polyacrylamide into 5 main subunits. 6. 6. When the upper band of the gradient was treated with phenol-formic acid-water and separated by gel electrophoresis in polacrylamide, only one of the 5 main subunits was obtained. This was characteristic of cytoplasmic membrane. 7. 7. The upper band of the gradient could be isolated from cultures grown under anaerobic light conditions, from cultures grown aerobically in the dark and from the carotenoid-free mutant strain of R. rubrum M 46, which forms only few chromatophores. 8. 8. The results indicate that the upper band of the Ficoll gradient consists mainly of mechanically disrupted cytoplasmic membrane. 9. 9. Morphogenetic relationships between cytoplasmic membrane and chromatophores are discussed.