X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. II. Comparison of diffraction patterns of photosynthetic units from various purple bacteria

Comparative X-ray diffraction studies, in conjunction with infrared absorption spectroscopy, were performed on chromatophores isolated from various purple photosynthetic bacteria in order to achieve a better understanding of the molecular structure of the photosynthetic unit. Purple non-sulfur bacteria used were Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas palustris. Chromatophores of Chromatium vinosum, as a typical example of purple sulfur bacteria, were also investigated. The results were as follows. Distinct equatorial X-ray diffraction patterns were obtained from chromatophores of all the bacteria examined. They showed diffuse, continuous diffraction patterns having several maxima, and the patterns are evidently distinguished from those of either crystalline or amorphous material. The pattern indicates that the photosynthetic unit in the chromatophore has a highly organized molecular structure in the plane of the membrane. Bacteria whose major photosynthetic pigment is bacteriochlorophyll alpha can be categorized in three groups from the viewpoint of near infrared absorption spectra. X-ray diffraction patterns are also grouped accordingly, although the differences are minimal and the patterns display common features. In other words, the bacteriochlorophyll forms, which are bacteriochlorophyll-protein complexes exhibiting different near-infrared absorption spectra, show different X-ray patterns: the molecular structure of photosynthetic units is closely related to the state of pigment in each complex, although the "X-ray" molecular structure is mainly concerned with the arrangement of constituent protein molecules at the present resolution, whereas the "spectroscopic" structure reflects the local environment of pigment.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. III. Basic structure of the photosynthetic unit and its relation to other bacteriochlorophyll forms

We have performed X-ray diffraction studies on photosynthetic units of Rhodospirillum rubrum and solubilized *B800 + B890 complex from chromatophores of Chromatium vinosum, to investigate the homology of their molecular structures. The native chromatophores of Chromatium vinosum, which contain other bacteriochlorophyll forms, were examined by an X-ray diffraction technique, in order to assess the interactions between the complexes as well as the molecular structures of the bacteriochlorophyll forms. The subchromatophore particles, solubilized by Triton X-100 from cells of Chromatium vinosum, exhibit a major absorption maximum at 881 nm and a minor one at 804 nm, consisting of bacteriochlorophyll form *B800 + B890. The near-IR absorption spectrum of the particle is very similar to that of chromatophores of Rhodospirillum rubrum although the major absorption maximum is shifted slightly. The X-ray diffraction pattern of the subchromatophore particles is very similar to that of chromatophores of Rhodospirillum rubrum. Thus, the subchromatophore particles are considered to be the "photoreaction unit" of Rhodospirillum rubrum. Since the bacteriochlorophyll form, *B800 + B890, is common in the purple bacteria, it is strongly suggested that the photoreaction unit is the basic and common structure existing in the photosynthetic units of purple bacteria. Chromatium vinosum cells exhibit different near-IR absorption spectra, depending on the culture media and also on the intensity of the illumination during culture. The chromatophores from these cells give different equatorial X-ray diffraction patterns. These patterns are much broader than that of solubilized subchromatophore particles, though they have common features. Thus, the molecular structures in the photosynthetic units are different, depending on their constituent bacteriochlorophyll forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane proteins of Rhodopseudomonas spheroides IV. Characterization of chromatophore proteins

Less than 5% of the protein isolated from Rhodopseudomonas spheroides chromatophores (designated Fraction P₁) is insoluble in 2-chloroethanol. Electrophoresis of these proteins on dodecyl sulphate-polyacrylamide gels reveals a gel pattern similar to those obtained from anaerobic and aerobic cell envelope proteins. Chromatophore P₁ is shown to be part of the chromatophore structure and its presence in the chromatophore is not due to contamination from the cytoplasmic membrane.Preparative dodecyl sulphate-polyacrylamide gel electrophoresis was performed to purify chromatophore P_ll proteins, which comprise 95% of the total chromatophore protein. These proteins contain approximately 60–65 mole% non-polar amino acids. Comparison studies of the amino acid compositions, tryptic and chymotryptic maps, molecular weights, and antigenic reactivity of chromatophore proteins demonstrate the existence of protein heterogeneity in chromatophores. These investigations lead us to suggest that chromatophore-specific proteins do not appear in other particulate or soluble fractions derived from either aerobic or anaerobic-grown cells.     

Structural changes accompanying the irreversible oxidation of the chromatophore membrane from Rhodospirillum rubrum G9

The structure of the chromatophore membrane of the carotenoid-free mutant Rhodospirillum rubrum G9 and the effect of irreversible photooxidation upon this structure have been investigated using several physical techniques. Native chromatophore membranes undergo endothermic transitions in two temperature regions; at temperatures of about 0°C a broad reversible transition and between approx. 50 and 90°C several endothermic transitions are observed which are irreversible. The first transition can be assigned to the gel-to-liquid crystal transition of the lipid bilayer present in chromatophores; the irreversible one is attributed to changes mainly in the quarternary and possibily tertiary structure of membrane proteins. CD measurements showed that heating of chromatophores up to 70°C has no effect upon the protein secondary structure. Photooxidation has little effect on the structure and dynamics of the lipid bilayer in the chromatophore membrane. The order (or average conformation) of both the lipid polar groups and the hydrocarbon chains is hardly changed. However, the lipid phase transition is dramatically broadened and the protein-associated endothermic transitions are greatly reduced. This indicates that the major effect of photooxidation is upon lipid-protein and protein-protein interactions. Electron microscopy studies support this interpretation. It can be shown that the dense and regular packing of protein particles observed in the chromatophore membrane is lost as an effect of photooxidation. Instead, randomly distributed particles of varying size and shape are seen. These results are interpreted to mean that pigment-protein interactions are responsible for maintaining the native long-range order in the chromatophore membrane of R. rubrum G9. Destruction of the pigments by photooxidation leads to irreversible protein dissociation which in turn is followed probably by random protein reaggregation.     

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides.

Triton extracts of intracytoplasmic photosynthetic membranes (chromatophores) purified from Rhodopseudomonas sphaeroides were subjected to crossed immunoelectrophoresis with antiserum raised in rabbits to purified chromatophores. A total of 31 immunoprecipitates was visualized; 2 of the immunoprecipitates were identified as reduced nicotinamide adenine dinucleotide (EC 1.6.99.3) and L-lactate dehydrogenases by enzyme staining techniques. Reaction with a monospecific antiserum identified the photochemical reaction center. Photopigments were associated with a major precipitate in the pattern which was identified on the basis of immunological identity as light-harvesting bacteriochlorophyll a . protein complex. These results provide the basis for a detailed structural and functional analysis of the chromatophore membrane by crossed immunoelectrophoresis.     

The dermal chromatophore unit

Rapid color changes of amphibians are mediated by three types of dermal chromatophores, xanthophores, iridophores, and melanophores, which comprise a morphologically and physiologically distinct structure, the dermal chromatophore unit. Xanthophores, the outermost element, are located immediately below the basal lamella. Iridophores, containing light-reflecting organelles, are found just beneath the xanthophores. Under each iridophore is found a melanophore from which processes extend upward around the iridophore. Finger-like structures project from these processes and occupy fixed spaces between the xanthophores and iridophores. When a frog darkens, melanosomes move upward from the body of the melanophore to fill the fingers which then obscure the overlying iridophore. Rapid blanching is accomplished by the evacuation of melanosomes from these fingers. Pale coloration ranging from tan to green is provided by the overlying xanthophores and iridophores. Details of chromatophore structure are presented, and the nature of the intimate contact between the chromatophore types is discussed.     

Assessment of Rhodopseudomonas sphaeroides chromatophore membrane asymmetry through bilateral antiserum adsorption studies.

The asymmetric structure of the Rhodopseudomonas sphaeroides chromatophore membrane was examined in detail by crossed immunoelectrophoresis techniques. Because these methods are quantitative and allow increased resolution and sensitivity, it was possible to analyze simultaneously the relative transmembrane distribution of a number of previously identified antigenic components. This was demonstrated by analysis of immunoglobulin samples that were adsorbed by preincubation with either isolated chromatophores or osmotically protected spheroplasts. The photochemical reaction center, the light-harvesting bacteriochlorophyll a-protein complex, the L-lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3) were found to be exposed on the chromatophore surface (cytoplasmic aspect of the membrane within the cell). Other antigenic components were found to be exposed on the surface of spheroplasts (periplasmic aspect of the in vivo chromatophore membrane). Antigens with determinants expressed on both sides of the chromatophore membrane were also identified. Charge shift crossed immunoelectrophoresis confirmed the suggested amphiphilic character of the pigment-protein complexes and identified several additional amphiphilic membrane components.     

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.     

Immunocytochemical ultrastructural analysis of chromatophore membrane formation in Rhodospirillum rubrum.

An immunocytochemical ultrastructural study of Rhodospirillum rubrum cultured under semiaerobic conditions was conducted to correlate the localization of functional components with membrane formation. R. rubrum is a facultatively phototrophic organism. Under reduced oxygen, this bacterium forms an intracytoplasmic chromatophore membrane that is the site of the photosynthetic apparatus. Immunogold techniques were used to localize intracellular protein antigens associated with the photosynthetic apparatus. Antibody, demonstrated by immunoblotting to be specific for the reaction center and light-harvesting photochemical components, was conjugated to colloidal gold particles and used for direct immunolabeling of fixed, sectioned specimens. Membrane invaginations appeared by 4 h after transition to induction conditions, and mature chromatophore membrane was abundant by 22 h. The occurrence of chromatophore membrane was correlated with bacteriochlorophyll a content and the density of the immunolabel. In uninduced (aerobic) cells and those obtained from cultures 0.5 h posttransition, the immunogold preferentially labeled the peripheral area of the cell. In contrast, in cells obtained after 22 h of induction, the central region of the cell was preferentially immunolabeled. These findings provided immunocytochemical evidence supporting the hypothesis that the chromatophore membrane is formed by invagination of the cytoplasmic 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.     

The structure of the purple membrane from Halobacterium hallobium: analysis of the X-ray diffraction pattern.

An X-ray diffraction analysis of oriented specimens of the purple membrane from Halobacterium halobium shows that the protein and lipid components are packed in a P3 hexagonal lattice, with one protein molecule per asymmetric unit. The structure is made up of a single layer of the protein molecules, oriented vectorially in the same direction across the membrane.The presence of strong diffraction peaks equatorially centred at 10 Å, and axially at 5 Å and 1.5 Å, show that the protein molecules, which make up most of the mass of the membrane, are composed to a considerable extent of α-helices, 25 to 35 Å long, arranged roughly perpendicular to the plane of the membrane to form superhelical groupings of the “coiled-coil” type.The surface of the membrane is flat, with no bumps or dimples large enough to affect the X-ray pattern when the electron density of the suspending medium is altered. The phospholipids may be less exactly positioned in the lattice than the protein, since the presence of uranyl acetate, which is expected to co-ordinate with the acidic phosphate groups, produces intensity changes only at low resolution.     

Probing the topology of proteins in the chromatophore membrane of Rhodospirillum rubrum G-9 with proteinase K

All the major membrane proteins of isolated chromatophore vesicles are eventually degraded upon incubation with the unspecific proteinase K. These proteins must therefore be exposed at least partially or temporarily on the cytosolic surface of the membrane which is exclusively accessible to the proteinase in intact chromatophore vesicles. That the vesicles are intact during the incubation with proteinase is demonstrated by the finding that cytochrome c₂, which is located in the interior of the vesicles, is protected from proteolytic attack. The degree of degradation of the various chromatophore proteins and the time taken for degradation differ characteristically. From the changes in intensity of the gel bands during the course of digestion it appears that reaction center subunit H is digested first, much faster than are subunits M and L. The near-infrared absorption spectrum of the chromatophores changes only after proteolytic degradation of these two pigment-carrying subunits. Fading of the band of the light-harvesting polypeptide is evident only after prolonged incubation. It seems that this is the most stable component of the chromatophore membrane. The light-harvesting polypeptide appears to be somewhat shortened eventually, leaving the protein conformation necessary for holding the pigments unchanged, as shown by the absorption spectrum. The possible topology of these major membrane components is discussed in the light of these findings.     

Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.     

Localization of photosynthetic reaction centers by antibody binding to chromatophore membranes from Rhodopseudomonas spheroides strain R26

Rabbit antiserum against highly purified reaction center preparations was shown to react specifically with a single component of chromatophore membranes from Rhodopseudomonas spheroides strain R-26. The conjugate of purified gamma globulin and ferritin prepared with toluene diisocyanate was used to determine the localization of reaction centers in the chromatophore membranes. Virtually no antibody was bound by intact membranes. After removing the 9 nm ATPase from these membranes by dilute EDTA treatment, a considerable amount of antibody was bound to the exposed outer membrane surface. The reaction center binding sites were estimated to be uniformly distributed with approx. 1 reaction center per 200 nm² of membrane surface. These results indicate that the reaction centers are located near the outer membrane surface but below the ATPase particles. Since the distribution of reaction centers and particles on rough faces seen by freeze-fracture electron microscopy are similar, it is suggested that the freeze-fracture particle may be a complex of a reaction center and other electron transfer components localized within the hydrophobic region of the membrane.     

Effects of surface potential and membrane potential on the midpoint potential of cytochrome c-555 bound to the chromatophore membrane of Chromatium vinosum

The values of midpoint potential (Em) of cytochrome c-555 bound to the chromatophore membranes of a photosynthetic bacterium Chromatium vinosum was determined under various pH and salt conditions. After a long incubation at high ionic concentrations in the presence of carbonylcyanide m-chlorophenylhydrazone, which was added to abolish electrical potential difference between the inner and outer bulk phases of chromatophore, the Em value was almost constant at pH values between 4.0 and 8.4. With the decrease of salt concentration, the pH dependence of the Em value became more marked. Under low ionic conditions, Em became more positive with the decrease of pH. Addition of salt made the value more positive or negative at pH values higher or lower than 4.5, respectively. Divalent cation salts were more effective than monovalent cation salts in producing the positive shift of Em at pH 7.8. The Em value became more positive when the electrical potential of the inner side of the chromatophore was made more positive by the diffusion potential induced by the K+ concentration gradient in the presence of valinomycin. These results were explained by a change of redox potential at the inner surface of the chromatophore membrane, at which the cytochrome is assumed to be situated, due to the electrical potential difference with respect to the outer solution induced by the surface potential or membrane potential change. The values for the surface potential and the net surface charge density of the inner surface of the chromatophore membrane were estimated using the Gouy-Chapman diffuse double layer theory.     

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.     

Electrical potential changes in the surface and the central region of chromatophore membranes of photosynthetic bacteria detected by the absorbance changes of ethidium and carotenoid

(1) Changes of local intramembrane electrical field in the surface and central region of the chromatophore membrane during energization were studied both by the measurement of absorbance changes of ethidium, a monovalent cationic dye, and of carotenoid, the intrinsic probe of electrical field. (2) Binding of ethidium to the chromatophore membrane of Rhodopseudomonas sphaeroides was found to be dependent on the energization of membrane as well as on the ionic condition of the medium. The dye was released from the membrane when salt was added to the suspension, indicating the electrostatic interaction between the positive dye and the net negative membrane surface. The result was explained by the surface-potential dependent distribution of the dye to the membrane surface, as seen with other charged dyes (Masamoto, K., Matsuura, K., Itoh, S. and Nishimura, M. (1981) Biophys. Acta 638, 108–115). (3) Energization of chromatophores by flash-light-induced absorbance change of ethidium showing a similar difference spectrum to that induced by the addition of salts. The release of ethidium by a single turn-over flash of saturating intensity was estimated to be 0.22 ethidium per reaction center. Addition of ethidium (at 200 μM) slightly affected the flash-induced absorbance change of carotenoid which responds to the intramembrane electricalfield change, indicating a low-membrane permeability of the dye. The extent of the absorbance change of ethidium was linear to that of carotenoid, and was abolished in the presence of valinomycin plus K⁺. However, the rise and decay kinetics of the absorbance change of ethidium was different from that of carotenoid. (4) These absorbance changes of ethidium and carotenoid can be explained by a model in which ethidium responds to the potential changes in the surface region and carotenoid in the central hydrophobic region of the chromatophore membrane.     

Xanthophores in chromatophore groups of the premigratory neural crest initiate the pigment pattern of the axolotl larva

Summary The barred pigment pattern (Lehman 1957) of the axolotl larva is best observed from stage 41 onwards, where it already consists of alternating transverse bands of melanophores and xanthophores along the dorsal side of the trunk. The present study investigateswhen the two populations of neural crest derived chromatophores, melanophores and xanthophores become determined andhow they interact to create the barred pigment pattern. The presence of phenol oxidase (tyrosinase) in melanophores (revealed by dopa incubation) and pteridines in xanthophores (visualized by fluorescence) were used as markers for cell differentiation in order to recognize melanophores and xanthophores before they became externally visible. It was found that melanophores and xanthophores were already determined in the premigratory neural crest, at stages 30/31 and 35–36, respectively. Between stages 35–36 and 38 they were arranged in a prepattern of several distinct, mixed chromatophore groups along the dorsal trunk, morphologically correlated in the scanning electron microscope with humps on the original crest cell string. While the occurrence of xanthophores was restricted to the chromatophore groups and around them, melanophores were already uniformly distributed in the dorsolateral flank area, having migrated from trunk neural crest portions including the groups. The bar component of the pigment pattern was subsequently initiated by xanthophores, which caused melanophores in and around the chromatophore groups to fade or become invisible. The barred pattern was established by the formation of alternating clusters of like cells, melanophores and xanthophores.     

Effects of exogenous guanosine on chromatophore differentiation in the axolotl

Guanosine is shown to dramatically alter the pigment phenotype of axolotls by suppressing melanization and enhancing the biosynthesis and deposition of purine-derived pigments. Phenotypic changes caused by guanosine are manifested by altered chromatophore differentiation patterns such that few black pigment cells (melanophores) differentiate (and those that do are punctate and necrotic in appearance), whereas the development of yellow (xanthophore) and reflecting (iridophore) pigment cells is enhanced. Mechanisms for changes in chromatophore differentiation, and thus pattern formation, are discussed, including the possibility that pigment cells may undergo transdifferentiation in vivo.     

Localisation of the subunits of the photosynthetic reaction centers in the chromatophore membrane of Rhodospirillum rubrum.

Reaction centers were isolated with the detergent lauryl dimethyl amine oxide from chromatophore membranes of Rhodospirillum rubrum. The subunit composition of these reaction centers is similar to the one obtained from Rhodopseudomonas spheroides: three subunits with the molecular weights of 21 000, 24 000 and 29 000. Reaction centers prepared from chromatophores labeled with 131I were heavely labeled in their large subunit (H). The smaller subunits (L and M) contained only little label. Sonication during labeling yielded a slightly higher incorporation of 131I in subunit H compared to the smaller ones. It is concluded that the H protein is largely exposed at the cytoplasmic side of the membrane but might also be accessible for iodination on the inside of the membrane while the L and M proteins are almost completely embedded in the membrane. Iodination of spheroplasts results in only a slight binding of 131I to chromatophores and reaction centers.