Heterogeneous profile in thermal denaturation of DNA preparation from the non-sulfur purple photosynthetic bacterium Rhodopseudomonas spheroides

Thermal denaturation profiles of DNA preparations from fourstrains of Rhodopseudomonas spheroides were comparatively studied.All the melting curves in 0.1 ? SSC displayed Tm at 82 ?0.5?and a faint bimodal transition near 78?. The differential meltingrates as a function of temperature exhibited several peaks,suggesting that inter- or intra-molecular structural heterogeneitymay be present in R. spheroides DNA. ¹ Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Japan. (Received February 15, 1975; )     

Light-induced absorbance change of carotenoid in chromatophores of photosynthetic bacterium, Rhodopseudomonas spheroides

The nature of the light-induced absorbance change of carotenoid,spheroidene, was investigated with the chromatophores of Rhodopseudomonasspheroides. The experimental results indicate that the changedoes not represent an oxidation-reduction reaction of the carotenoid,but is caused by a change in the state of the chromatophoresclosely related to the high energy state of the photophosphorylation.Since the change almost vanishes at liquid nitrogen temperature,it probably does not represent a primary photochemical reactionin the chromatophores. The values of the quantum yield for thechange of carotenoid were above unity ; 2.5 on an avera (Received November 20, 1969; )     

Isolation and fractionation of the photosynthetic membranous organelles from Rhodopseudomonas spheroides

Molecular sieve chromatography and sucrose gradient centrifugation were used to prepare large quantities of purified chromatophores from Rhodopseudomonas spheroides. Electron micrographs of these chromatophores revealed that the final preparations were very homogeneous and free of non-chromatophore particulate material. As an additional check on purity, (14)C-l-phenylalanine-labeled aerobic cells, devoid of chromatophores, were mixed with unlabeled photosynthetic cells. The resulting preparation contained less than 1% of the radioactivity, originally located in non-chromatophore protein. The purified chromatophores were solubilized in 2-chloroethanol and separated into two fractions. Fraction P(1) contained 3 to 5% of the total chromatophore protein and could be resolved into 10 electrophoretic components. The second fraction, P(II), contained five electrophoretic components. One of these components had associated with it all of the pigment and phospholipid present in P(II). Preliminary immunochemical studies on these fractions are also reported.     

Electron acceptors in photosynthetic reaction centers from Rhodopseudomonas spheroides]

Using optical differential spectroscopy and EPR, a parallel study of light-induced electron transfer between the primary (X1) and secondary (X2) quinone-like acceptors in the preparations of reaction centers (RC) isolated from bacterial chromatophore membranes with sodium dodecyl sulfate was carried out. The data from direct measurements of the rate constant temperature dependence for the interaction between light-reduced X1 and X2 (KX1X2) are in good agreement with the data calculated from the kinetic analysis of dark reduction of photooxidized bacteriochlorophyll RC on the acceptors X1 and X2 (KX1X2 = 2.10(-1)S at 20 degrees; Ea = 11,8 kcal.mol-1 within the temperature range of 20 degrees-- -20 degrees). This evidence proves the efficiency of the previously used approach /1, 2/ for the evaluation of the X1-X2 interaction. The method proposed was used for a kinetic analysis of a low-temperature electron transfer from X1 to X2 in RC isolated with lauryldimethylaminoxide (KX1X2 = 2,3.10(2) S-1 at 20 degrees; Ea = 5,5 kcal.mol-1 within the temperature range of 10 degrees-- --70 degrees).     

Isolation and characterization of cytochrome C1 from photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

Cytochrome c1 of photosynthetic bacterium R. sphaeroides R-26 has been purified from isolated cytochrome b-c1 complex to a single polypeptide, using a procedure involving Triton X-100 and urea solubilization, calcium phosphate column chromatography and ammonium sulfate fractionation. The purified protein contains 30 nmoles heme per mg protein and has an apparent molecular weight of 30,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis. Bacterial cytochrome c1 is soluble in aqueous solution in the absence of detergent and has spectral characteristics similar to mammalian cytochrome c1. The amino acid compositions of these two proteins, however, are not comparable.     

Lipid-protein associations in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid-protein interactions were examined in chromatophores isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides using lipid spin-labels. The chromatophores contain fluid bilayer and a significant amount of lipid immobilized by membrane proteins. For a typical preparation of cells grown under 600 ft-c illumination, 59% of the spin-labeled fatty acids were bound. Essentially the entire length of the 18-carbon fatty acid chain was immobilized, judging from results obtained with the spin-label at the 7, 12, and 16 positions. The amount immobilized varies directly with the bacteriochlorophyll content of the chromatophore material, suggesting that a significant fraction of the lipid spin-labels is immoblized on the hydrophobic surfaces of the chlorophyll-binding proteins. Changing the lipid spin-label head group from a negatively charged carboxyl group to a positively charged quarternary amine greatly decreased the amount of immobilized lipid. The changes in immobilized lipid with light level and polar head group suggest that the anntenna bacteriochlorophyll-binding proteins preferentially associate with negatively charged lipids.     

Purification and characterization of a catalase-peroxidase from the photosynthetic bacterium Rhodopseudomonas capsulata

Catalase-peroxidase was isolated from aerobically grown Rhodopseudomonas capsulata. The enzyme resembles typical catalases in some of its physicochemical properties. It has an apparent molecular weight of 236,000 and is composed of four identical subunits. It shows a typical high spin ferric heme spectrum with absorption maxima at 403 and 635 nm and shoulders at 503 and 535 nm. Upon binding of cyanide, the enzyme is converted to the low spin state, as shown by the shift of the Soret maximum to 418 nm and the band at 532 nm. It has an isoelectric point at pH 4.5. The enzyme differs from typical catalases in also having a strong peroxidatic activity with dianisidine, pyrogallol, and diaminobenzidine as electron donors. Both the catalatic and the peroxidatic activities are similarly inactivated by treatment with 1 mM H2O2, heating to 50 degrees C, exposure to ethanol/chloroform, and photooxidative conditions. In contrast to typical catalases, but similarly to peroxidases, the enzyme is reduced by sodium dithionite. The pH optimum of the peroxidatic activity is 5-5.3 (in contrast to 6-6.5 of the catalatic activity). 50% of the apparent maximal activities are reached at 0.3 and 4.2 mM H2O2 for the peroxidatic and catalatic activities, respectively. Both enzymic activities are equally inhibited by cyanide, 50% inhibition being achieved with 2.2 X 10(-5) M KCN. Contrarily, the two activities differ in their response to hydroxylamine and azide. 50% inhibition of the catalatic activity is obtained with 1.5 X 10(-4) M azide or 2.15 X 10(-6) M hydroxylamine; 50% inhibition of the peroxidatic activity requires 7.3 X 10(-4) M azide or 7.8 X 10(-5) M hydroxylamine. The activation energies of the catalatic and the peroxidatic activities are 1.9 and 1.7 kcal/mol, respectively.     

Kinetics of photosynthetic membrane protein assembly in Rhodopseudomonas spheroides

Glycogen phosphorylase in cell-free extracts of Neurospora crassa is activated 10- to 15-fold by incubation with MgATP^2?. When the MgATP^2? is removed, the active form (a form) reverts to the inactive form (b form). The inactivation requires Mg²⁺ and is inhibited by NaF. The results confirm that Neurospora crassa glycogen phosphorylase exists in two interconvertible forms and strongly suggests that the interconversion is catalyzed by a kinase and phosphatase. The a form was partially purified. The enzyme has a molecular weight of 320,000. Uridine diphosphate glucose is a linear competitive inhibitor with respect to glucose-1-phosphate and a linear non-competitive inhibitor with respect to glycogen. Glucose-6-phosphate is a hyperbolic (partial) noncompetitive inhibitor with respect to all substrates in both directions. The b form of the enzyme in crude cell-free extracts is stimulated 2- to 3-fold by 5′-AMP. As the b form is purified, the 5′-AMP activation is diminished. The molecular weight of the partially purified “b” form was also 320,000.     

Inorganic nitrogen assimilation by the photosynthetic bacterium Rhodopseudomonas capsulata.

The photosynthetic bacterium Rhodopseudomonas capsulata lacks glutamate dehydrogenase and normally uses the glutamine synthetase/glutamate synthase sequence of reactions for assimilation of N2 and ammonia. The glutamine synthetase in cell-free extracts of the organism is completely sedimented by centrifugation at 140,000 X g for 2 h, is inhibited by L-alanine but not by adenosine 5'-monophosphate, and exhibits two apparent Km values for ammonia (ca. 13 muM and 1 mM).     

Plasmid rearrangements in the photosynthetic bacterium Rhodopseudomonas sphaeroides

Mu d1(Ap lac) was introduced into the photosynthetic bacterium Rhodopseudomonas sphaeroides 2.4.1. via the R-plasmid R751 in an attempt to isolate fusion derivatives involving photosynthetic operons. The selection system is potentially very powerful since R. sphaeroides is normally Lac negative. Among the exconjugants, photosynthesis-deficient mutants were recovered, some of which had elevated beta-galactosidase levels. Among the mutants examined, beta-galactosidase expression was linked exclusively to R751 . Many of the photosynthesis-deficient mutants were found to have alterations in their indigenous plasmids which apparently involved the exchange of DNA from one plasmid to another. Southern blot analysis revealed that there are extensive DNA sequences which are shared by the two plasmids that are involved in the rearrangements and that no exogenous DNA sequences appear to be involved. It was further discovered that plasmid rearrangement is a general phenomenon which can occur spontaneously in R. sphaeroides 2.4.1 and shows a high correlation with a photosynthesis minus phenotype.     

Nobel lecture. The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis.

In our lectures we first describe the history and methods of membrane protein crystallization, before we show how the structure of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis was solved. Then the structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Finally we draw conclusions on the structure of the photosystem II reaction centre from plants and discuss the aspects of membrane protein structure. Sections 1 (crystallization), 4 (conclusions on the structure of photosystem II reaction centre and evolutionary aspects) and 5 (aspects of membrane protein structure) were presented and written by H.M., Sections 2 (determination of the structure) and 3 (structure and function) by J.D. We have arranged the paper in this way in order to facilitate continuous reading.     

Bacteriophages of Rhodopseudomonas spheroides: Isolation and Characterization of a Rhodopseudomonas spheroides Bacteriophage

A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.     

Nucleotide exchange in membrane vesicles from the photosynthetic bacterium Rhodopseudomonas capsulata

Membrane vesicles (heavy chromatophores) prepared from the photosynthetic bacteria Rhodopseudomonas capsulata catalyze photophosphorylation of exogenous ADP and also take up [³H]ADP from the external medium. The rate of uptake depends on the concentration of external ADP reaching half-maximal velocity at 2.7 mm. The rate increases also with the increase in the concentration of internal ADP. Vesicles, preloaded with [³H]ADP release the radioactive nucleotide when ADP is included in the external medium. Regular chromatophores, which are inside-out membrane vesicles also take up [³H]ADP from the external medium when preloaded with ADP. These results are interpreted to indicate the existence of nucleotide transport across the cytoplasmic membrane of these bacteria which is catalyzed by an ADP exchange carrier.     

Primary structure of a high potential iron-sulfur protein from the purple non-sulfur photosynthetic bacterium Rhodopseudomonas gelatinosa.

The third amino acid sequence of a high potential iron-sulfur protein, that of the non-sulfur purple photosynthetic bacterium Rhodopseudomonas gelatinosa, has been determined. It consists of a single polypeptide chain of 74 amino acid residues, which is slightly smaller than the high potential iron-sulfur proteins from the sulfur purple bacteria Chromatium vinosum (85 residues) and Thiocapsa pfennigii (81 residues). The sequence of the gelatinosa protein is similar to the C. vinosum and T. pfennigii proteins with 38% and 37% identically matching residues, although six gaps are proposed for the comparison (the C. vinosum and T. pfennigii proteins have 44% identically matching residues out of 73 positions compared with only one 4-residue gap). Only 17 redisues, including the 4 cystein residues essential for binding the four-iron-sulfur chromophore, are invariant in the three known sequences. A discussion of the role of conserved residues in maintenance of the three-dimensional structure and in electron transport is presented.